CN113056479A - CD40 and CD40L conjugates in an adenoviral vaccine vehicle - Google Patents

Abstract

A cancer vaccine is provided that includes a recombinant nucleic acid encoding a self-activating chimeric signaling protein, and in particular a chimeric TNF family ligand-receptor protein, and a tumor associated antigen. In preferred embodiments, the cancer vaccine may further comprise a nucleic acid segment encoding an IL-15 superagonist. In addition, the cancer vaccine can be co-administered with genetically modified bacteria or yeast as an adjuvant, thereby increasing the payload expression of the cancer vaccine. Advantageously, cells expressing this combination of molecules will enhance the immune response against tumor cells. Compositions and methods are presented that allow for enhanced immune responses against vaccine compositions, and in particular recombinant adenoviral expression systems for use as therapeutic agents. Most preferably, the immunotherapeutic agent is administered such that the protein or nucleotide is co-localized with the therapeutic antigen, preferably via co-expression of the protein.

Description

CD40 and CD40L conjugates in an adenoviral vaccine vehicle

This application claims priority to co-pending U.S. provisional application serial No. 62/742,167 filed on day 10, month 5 of 2018 and No. 62/755,217 filed on day 11, month 2 of 2018.

Sequence listing

An ASCII text file of Sequence listing, named Sequence _ listing _ st25.txt, size 45KB, was created on 2019, 9, 19, electronically filed with the application via EFS-Web, and its contents are incorporated by reference in their entirety.

Technical Field

The present disclosure relates to cancer vaccines comprising recombinant nucleic acids encoding CD40/CD40L fusion proteins and tumor associated antigens, as well as compositions and methods for improved neoepitope-based immunotherapy. Certain specific disclosures relate to compositions comprising the above-described nucleic acids and/or fusion molecules, and methods of using these nucleic acids and/or fusion molecules to enhance immune responses against cancer therapies.

Background

The following description includes information that may be useful in understanding the present disclosure. Nothing herein is to be construed as an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is any publication specifically or implicitly referenced as prior art.

All publications identified herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

TNF family member receptors (e.g., CD40, 4-1BB, or OX40) and their respective ligands play a key role in the regulation of cellular and humoral immunity. For example, 4-1BB signaling, together with NK cell activation, increases antibody-dependent cellular cytotoxicity (ADCC) and interferon gamma (IFN- γ) secretion, while OX40 signaling is associated with T cell activation and differentiation. In other examples, a variety of immune cells express CD40, and antigen presenting cells (APCs, e.g., dendritic cells, macrophages, and B cells) also express CD 40. Among other effects, CD40L/CD40 is critical for activating and "permissioning" dendritic cells to trigger cytotoxic CD8+ T cells. Most typically, CD40 ligand (CD40L) expressed on CD4+ helper T cells engages CD40 on APCs, thereby inducing APC activation and maturation. CD40 licensed APCs induce activation and proliferation of antigen-specific CD8+ cytotoxic T cells. Notably, in the absence of CD40 signaling, CD8+ T cells and unlicensed APCs induce T cell anergy and trigger regulatory T cell formation, a mechanism by which tumors persist in mammals despite the presentation of other antigenic peptides.

Agonistic antibodies or soluble CD40L can be used to effectively trigger CD40 signaling (e.g., Int Rev Immunol [ international immunological review ]2012,31: 246-66). However, such methods are limited by systemic toxicity (e.g., J Clin Oncol [ J. Clin Oncol ]2007,25: 876-83; Science [ Science ]2012,331: 1612-16).

CD40 signaling efficacy is dependent on CD40 multimerization. The multi-trimer fusion construct of CD40L and gp100 tumor antigen can activate dendritic cells and improve survival in a B16-F10 melanoma DNA Vaccine model (see, e.g., Vaccine 201533 (38): 4798-806).

In WO 00/63395 a chimeric polypeptide is reported consisting of a CD40 signalling domain fused to a 50-100 amino acid spacer, which in turn is fused to a CD40L binding and trimerising domain.

Similarly, in WO 02/36769 a chimeric polypeptide consisting of a CD40 signalling domain fused to a type 2 receptor transmembrane domain, which in turn is fused to a CD40L binding and trimerising domain, is reported.

Neither WO 00/63395 nor WO 02/36769 reported a therapeutic effect in mice implanted with tumor cells transfected with these constructs.

In US 7,404,950 a chimeric protein consisting of a CD40 cytoplasmic domain fused to an FK506 ligand binding domain and a myristoylation membrane targeting domain is reported.

Fusion proteins with multimeric ligand binding regions and a CD40 portion lacking the extracellular domain are reported in US 8,999,949.

While such constructs may provide some increased activity in vitro, they tend to be antigenic when administered to a mammal.

Thus, while various means of modulating TNF family member receptor/ligand signaling are known in the art, all or almost all of these means suffer from one or more disadvantages. Thus, there remains a need to improve the modulation of TNF family receptor/ligand signaling.

In addition, immunotherapy targeting certain antigens shared by specific cancers elicits significant responses in some patients. Unfortunately, despite the dominant expression of the same antigen, many patients fail to respond to this immunotherapy. One possible reason for the failure of this response is that various immune effector cells may not be present in sufficient quantities, or may have been depleted. Moreover, intracellular antigen processing and HLA variability in patients may have resulted in insufficient antigen processing and/or display.

Some random mutations in tumor cells may give rise to unique tumor-specific antigens (neo-epitopes). Neoepitopes can provide unique, precise targets for immunotherapy. In addition, very small amounts of peptide may trigger cytolytic T cell responses (e.g., Sykulev et al (1996) Immunity, 4(6): 565-71). Moreover, the number of possible targets is relatively high due to the relatively high number of mutations in many cancers. In view of these findings, the identification of cancer neoepitopes as therapeutic targets has attracted considerable attention. Unfortunately, current data indicates that nearly all neoepitopes are unique to both the patient and the particular tumor, and fails to provide any specific indication as to which neoepitope is useful for a therapeutically effective immunotherapeutic.

However, even when filtering mutation types of neoepitopes (e.g., to determine missense or nonsense mutations), confirmed transcription of mutant genes, protein expression, and/or specific HLA binding (as described in WO 2016/172722), a durable and therapeutically effective immune response may still be elusive. For example, immunity may be prevented by inhibitory conditions in the tumor microenvironment. Furthermore, not all neo-epitopes trigger immune responses with the same intensity. Some new epitopes may be less immunogenic.

Although a variety of methods for the identification and delivery of neo-epitopes to a variety of cells are known, all of these methods have a number of disadvantages. Accordingly, there is a need for improved systems and methods for neoepitope selection and production to increase the likelihood of therapeutic response.

Disclosure of Invention

Disclosed herein are compositions, methods and uses of recombinant nucleic acids encoding chimeric proteins comprising a TNF family member ligand and a TNF family member receptor and encoding a tumor associated antigen. Genetically modified immune cells comprising such nucleic acids are also disclosed. Methods of treating cancer using such recombinant nucleic acids and/or genetically modified immune cells are also disclosed. For example, provided herein are recombinant expression cassettes comprising a promoter operably coupled to a recombinant nucleic acid. The recombinant expression cassette may be part of an RNA and/or viral expression vector. The recombinant expression cassette comprises a first nucleic acid segment encoding a chimeric protein having an extracellular portion of a TNF family member ligand coupled to its corresponding TNF family member receptor by a flexible linker and a second nucleic acid segment encoding a tumor associated antigen. Most typically, the extracellular portion of the TNF family member ligand is located N-terminally relative to the TNF family member receptor in the chimeric protein. Furthermore, preferably the flexible linker has between 4 and 50 amino acids and optionally comprises a (GnS) x sequence.

In certain embodiments, the recombinant expression cassette comprises a third nucleic acid segment encoding a leader peptide coupled to the N-terminus of the extracellular portion of CD40L (CD40 ligand). In such embodiments, the extracellular portion of CD40L may be the human extracellular portion of CD 40L. In some embodiments, the tumor associated antigen is selected from the group consisting of brachyury (brachyry), MUC1, and CEA. In other embodiments, the tumor-associated antigen is a patient-and tumor-specific neoepitope.

In certain preferred embodiments, the first and second nucleic acid segments are placed in the same reading frame. Alternatively, the first and second nucleic acid segments may be coupled via an IRES or 2A sequence.

In certain embodiments, the recombinant expression cassette may further comprise a fourth nucleic acid segment encoding an immunostimulatory cytokine. In such embodiments, the immunostimulatory cytokine may be an IL-15 superagonist (ALT803) coupled to at least one of IL-7 and IL-21.

In certain embodiments, the genetically engineered virus may comprise a recombinant expression cassette as described above.

In still other embodiments, a genetically modified immune cell can comprise a recombinant nucleic acid having first and second nucleic acid segments, and optionally third and fourth nucleic acid segments. The first nucleic acid segment encodes a chimeric protein having an extracellular portion of a TNF family member ligand coupled to its corresponding TNF family member receptor by a flexible linker. The second nucleic acid segment encodes a tumor associated antigen. Most typically, the extracellular portion of the TNF family member ligand is located at the N-terminus of the chimeric protein and the TNF family member receptor is located at the C-terminus of the chimeric protein. Furthermore, preferably the flexible linker has between 4 and 50, or between 8 and 50, or even more amino acids, and optionally comprises a (GnS) x sequence.

The genetically modified immune cells may be derived from dendritic cells, and more preferably, dendritic cells of the patient (allogeneic dendritic cells). In such embodiments, the patient's own dendritic cells can be obtained from the patient's blood and ex vivo amplified before and/or after genetic modification with the recombinant nucleic acid.

Preferably, the recombinant nucleic acid comprises a third nucleic acid segment encoding a leader peptide coupled to the N-terminus of the extracellular portion of CD 40L. In such embodiments, the extracellular portion of CD40L is a human extracellular CD40L moiety.

Also disclosed herein are methods of treating a patient having a tumor. The genetically engineered virus can be administered to a patient at a dose and on a schedule effective to treat the tumor. Most typically, the genetically engineered virus includes a recombinant nucleic acid having first and second nucleic acid segments. The first nucleic acid segment encodes a chimeric protein having an extracellular portion of a TNF family member ligand coupled to its corresponding TNF family member receptor by a flexible linker. The second nucleic acid segment encodes a tumor associated antigen. Most typically, the extracellular portion of the TNF family member ligand is located N-terminally relative to the TNF family member receptor in the chimeric protein. Furthermore, preferably the flexible linker has between 4 and 50 amino acids and optionally comprises a (GnS) x sequence.

In certain preferred embodiments, the recombinant nucleic acid comprises a third nucleic acid segment encoding a leader peptide coupled to the N-terminus of the extracellular portion of CD 40L. In such embodiments, the extracellular portion of CD40L may be an extracellular portion of human CD 40L. In some embodiments, the tumor associated antigen is selected from the group consisting of short-tail mutein, MUC1 and CEA. In other embodiments, the tumor-associated antigen is a patient-and tumor-specific neoepitope.

In certain preferred embodiments, the first and second nucleic acid segments are placed in the same reading frame. Alternatively, the first and second nucleic acid segments may be coupled via an IRES sequence. In addition, the recombinant nucleic acid may further comprise a fourth nucleic acid segment encoding an immunostimulatory cytokine. In such embodiments, the immunostimulatory cytokine may be an IL-15 superagonist (ALT803), alone or coupled to at least one of IL-7 and IL-21.

Optionally, the method may further comprise administering to the patient a checkpoint inhibitor and/or an IL-15 superagonist (ALT803), wherein the checkpoint inhibitor or ALT803 is coupled to at least one of IL-7 and IL-21. In addition, the method may comprise co-administering the genetically modified bacteria or the genetically modified yeast as an adjuvant to the genetically engineered virus. In such embodiments, the genetically modified bacteria may express endotoxin at levels insufficient to induce CD-14 mediated sepsis. In certain embodiments, the genetically modified yeast is a GI-400 series recombinant immunotherapeutic yeast strain.

Also disclosed herein is the use of genetically engineered viruses, recombinant expression cassettes, and/or genetically modified immune cells for the production of pharmaceutical compositions for treating patients with cancer or for treating patients with cancer.

In a particularly preferred aspect, a CD40L-CD40 fusion protein is constructed and expressed in APCs, wherein the fusion protein is capable of folding upon itself to transmit CD 40-mediated signals as if it were activated by a separate cell with CD40L (e.g., a CD4+ T cell). Similarly, in further contemplated aspects, the 4-1BB ligand/4-1 BB and Ox40L/Ox40 fusion proteins can be expressed in suitable immune competent cells.

Described herein is a chimeric protein comprising, in sequence from N-terminus to C-terminus, the extracellular portion of CD40L coupled to CD40 by a flexible linker. In certain embodiments, the chimeric protein further comprises a leader peptide coupled to the N-terminus of the extracellular portion of CD 40L.

In certain preferred embodiments, the extracellular portion of CD40L is a human extracellular portion, and CD40 is human CD 40. In certain preferred embodiments, the flexible linker has between 4 and 25 or between 8 and 50 amino acids (e.g., including (G)_nS)_xMotif, wherein n and x are independently between 1 and 5). Most typically, CD40 lacks a signal sequence compared to the full-length sequence. In certain embodiments, the chimeric protein can have a sequence according to any one of SEQ ID NOs 1-10.

Also disclosed herein are recombinant expression cassettes comprising a promoter operably coupled to a segment encoding a chimeric protein as described above. The recombinant expression cassette may further comprise a second segment encoding a cytokine and/or at least a portion of a peptide selected from the group consisting of: tumor Associated Antigens (TAAs), Tumor Specific Antigens (TSAs), tumor specific neo-epitopes, and combinations thereof. The recombinant expression cassette may be an RNA, or may be part of a viral expression vector (which may or may not be encapsulated).

Described herein are recombinant cells transfected with a recombinant expression cassette as described herein. In certain embodiments, the cell is an APC (e.g., a dendritic cell), and/or the cell is transiently transfected.

Also described herein are methods of enhancing an immune response against an antigen. These methods comprise transfecting an APC with a nucleic acid construct comprising a recombinant expression cassette as described herein and contacting the transfected cell with or expressing the antigen in the transfected cell. Following contact or expression, the transfected cells are then contacted with CD4+ T cells and/or CD8+ T cells.

As a non-limiting example, tumor and patient specific neo-epitopes or at least a portion of a Tumor Associated Antigen (TAA) or Tumor Specific Antigen (TSA) can be used as antigens for the above-described methods. Transfection may be performed ex vivo, and contact may be performed in vivo. Thus, the response to the antigen may be an immune response against a tumor or a virus (e.g., HIV) in the individual.

Also disclosed herein are methods of treating a tumor in an individual. These methods comprise transfecting an APC of the individual with a recombinant expression cassette as described herein and contacting the transfected cell with or expressing the tumor antigen in the transfected cell. After contacting or expressing, the transfected cells are then contacted with CD4+ T cells and/or CD8+ T cells of the subject.

As previously described, transfection may be performed ex vivo, and contact may be performed in vivo. Furthermore, the tumor antigen may be at least a part of a tumor and patient specific neoepitope or a TAA or TSA. In a preferred aspect, the APC is a dendritic cell and the recombinant expression cassette is part of an mRNA or adenovirus.

In certain embodiments, the chimeric proteins and/or recombinant cells as described herein can be used to treat cancer or viral infection.

Various immunotherapeutic compositions and methods are described herein. In particular, recombinant viral expression systems are described in which an adjuvant polypeptide is encoded with a plurality of selected neo-epitopes (typically in the form of rationally designed polypeptides with trafficking signals) to increase antigen processing and presentation and maximize therapeutic effect.

Methods of producing expression vectors and expression vectors for enhancing immunotherapy are described herein. These methods include constructing a recombinant nucleic acid having a sequence encoding a polyepitope operably linked to a first promoter to drive expression of the polyepitope, and further encoding an adjuvant polypeptide operably linked to a second promoter to drive expression of the adjuvant polypeptide. Most preferably, the polyepitope comprises a trafficking element that directs the polyepitope to a subcellular location (e.g., cytoplasm, recirculating endosomes, sorting endosomes, lysosomes, or extracellular membrane). Additionally or alternatively, the trafficking element may direct the multiple epitopes to the extracellular space. The polyepitope may also include a plurality of filtered new epitope sequences.

In certain embodiments, the adjuvant polypeptide is calreticulin or HMGB1, or a portion of calreticulin or HMGB1 that has adjuvant activity. The first and/or second promoter may be a constitutively active promoter or an inducible promoter (e.g., inducible by hypoxia, IFN-. gamma., or IL-8). Suitable trafficking elements include, but are not limited to, cleavable ubiquitin, non-cleavable ubiquitin, CD1b leader, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

Most typically, the filtered new epitope sequences are filtered by comparing tumors from the same patient to the matching normal sequences. The sequence may also be filtered to have a binding affinity for MHC complexes of ≧ 200 nM. Furthermore, the filtered neoepitope sequences may be arranged within the polyepitope such that the polyepitope has a potential for the presence of a hydrophobic sequence or signal peptide, and/or has a hydrophobic sequence or signal peptide intensity below a predetermined threshold.

In certain embodiments, the filtered neoepitope sequence binds to MHC-I, and the trafficking element directs the polyepitope to the cytoplasm or proteasome. In certain embodiments, the filtered novel epitope sequences bind to MHC-I, and the trafficking element directs the polyepitope to a recirculating endosome, a sorting endosome, or a lysosome. In certain embodiments, the filtered neoepitope sequence binds to MHC-II, and the trafficking element directs the polyepitope to a recirculating endosome, a sorting endosome, or a lysosome. In addition, the recombinant nucleic acid can further comprise a sequence encoding a second polyepitope, wherein the second polyepitope comprises a second trafficking element that directs the second polyepitope to a different subcellular location, and wherein the second polyepitope comprises a second plurality of filtered neo-epitope sequences. In this case, at least some of the plurality of filtered new sequence of table bits and some of the second plurality of filtered new sequence of table bits may be the same.

As disclosed herein, the recombinant nucleic acid can further comprise a sequence encoding at least one of: costimulatory molecules (e.g., OX40L, 4-1BBL, CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3), immunostimulatory cytokines (e.g., IL-2, IL-12, IL-15 superagonists (ALT803), IL-21, IPS1, and LMP1), and/or proteins that interfere with or down-regulate checkpoint inhibition (e.g., antibodies or antagonists to CTLA-4, PD-1, 1 receptors, 2B4, or CD 160).

Suitable expression vectors include adenovirus expression vectors, yeast expression vectors, and bacterial expression vectors deleted for the E1 and E2b genes. Recombinant viruses, yeasts and bacteria comprising the expression vectors set forth herein are described. Also described herein are pharmaceutical compositions comprising recombinant viruses, yeast or bacteria carrying the recombinant expression vectors. Also disclosed herein is the use of the expression vector for the treatment of cancer and/or the preparation of a vaccine composition for the treatment of cancer.

Described herein are methods of treating an individual. These methods comprise administering a vaccine composition comprising an expression vector as set forth herein, wherein the vaccine is administered under conditions effective to simultaneously expose dendritic cells of the individual to at least a portion of the polyepitope and at least a portion of the adjuvant polypeptide.

Alternatively or additionally, described herein are methods of improving an immune response to cancer. These immunotherapies in an individual include administering a cancer vaccine composition to a tumor of the individual and co-administering an adjuvant polypeptide, ATP, or ATP analog to the tumor substantially simultaneously (i.e., when the cancer vaccine composition is present in a measurable amount in the patient).

The cancer vaccine composition may comprise a recombinant adenovirus, a recombinant yeast, or a recombinant bacterium, and/or comprise or encode a tumor neoepitope of a patient. As a non-limiting example, the cancer vaccine composition may be administered directly to a tumor. Suitable adjuvant polypeptides include, but are not limited to, calreticulin or a portion thereof having adjuvant activity, or HMGB1 or a portion thereof having adjuvant activity. In certain embodiments, the adjuvant is a non-hydrolyzable ATP analog. The adjuvant may be injected directly into the tumor.

Various objects, features, aspects and advantages of the technology disclosed herein will become more apparent from the following detailed description and the accompanying drawings.

Drawings

Fig. 1 depicts several views of the predicted structure of an exemplary fusion protein.

Fig. 2 depicts the results of cells expressing exemplary fusion proteins.

Figure 3 demonstrates that these constructs are operable in different species (murine).

FIG. 4 depicts the secretion of IL-8 in selected cell lines.

Figure 5 demonstrates that the constructs are operable in different species.

Fig. 6 depicts surface expression in 293T cells.

Fig. 7 depicts surface expression in B16F10 cells.

FIG. 8 compares 293T cells transfected with CD40 and subsequently stimulated with soluble CD40L, versus CD40-CD40L fusions.

Figure 9 depicts the production of 293T (human) and B16F10 (mouse) cytokines from cells transfected with human/mouse constructs.

Fig. 10A illustrates an exemplary dimer of chimeras comprising hIL7 and IL 21. Fig. 10B illustrates an exemplary dimer of chimeras comprising mIL7 and IL 21. Fig. 10C illustrates an exemplary dimer of chimeras comprising hIL 21. Fig. 10D illustrates an exemplary dimer of chimeras including hIL 7. Fig. 10E illustrates an exemplary dimer of chimeras comprising hIL18 and IL 12. Fig. 10F illustrates an exemplary dimer of chimeras including hIL 18.

FIG. 11 is a schematic illustration of various new bit arrangements.

FIG. 12 is an exemplary partial schematic diagram for selecting a preferred new bit arrangement.

Prior art FIG. 13 is a schematic of cytoplasmic antigen processing and MHC-I presentation.

Prior art FIG. 14 is a schematic of lysosomal and endosomal antigen processing and MHC-II presentation.

FIG. 15 is a schematic of a recombinant adenovirus expression cassette for use in a cancer vaccine.

Detailed Description

As disclosed herein, the immune response against tumor cells can be modulated in a desired direction (i.e., enhanced or attenuated) by interfering with CD40 signaling in the APC. An immune response against tumor cells can be significantly enhanced by inducing APCs to express one or more TAAs. Vaccine compositions that induce expression of chimeric proteins and TAAs in APCs can treat tumors that express the TAAs. Thus, described herein are recombinant expression cassettes comprising a nucleic acid sequence encoding a TAA and a chimeric protein that modulates a CD40 signaling event.

As used herein, "tumor" refers to and is used interchangeably with: one or more cancer cells, cancer tissue, malignant tumor cells, or malignant tumor tissue, which may be located or found in one or more anatomical locations of a human body. As used herein, "binding" may be used interchangeably with "identifying" and/or "detecting" to convey having a value equal to or less than 10^{- 3}M、10^-4M、10^-5M、10^-6M is equal to or less than 10^-7Affinity of M (K)_D) The interaction between two molecules. As used herein, "providing" means and includes any act of manufacturing, producing, placing, enabling or preparing for use.

Chimeric protein (CD40/CD40L)

As used herein, "chimeras" are interchangeable with "chimeric proteins". The chimeric proteins disclosed herein preferably comprise a TNF family ligand (preferably, the extracellular portion of the ligand) and its corresponding TNF family receptor. These chimeras can mimic or induce signaling cascades in cells. Exemplary TNF family ligands and corresponding receptors include CD40/CD40L, 4-1BB/4-1BBL, and OX-40/OX-40L. Since these proteins share common structural motifs and activation patterns, the CD40/CD40L examples and examples presented herein are equally applicable to 4-1BB/4-1BBL and OX-40/OX-40L.

Chimeric proteins with the extracellular portion of a TNF family member ligand and its corresponding TNF family member receptor can self-activate to initiate signal transduction. For example, a chimeric protein having the extracellular domain of CD40L and CD40 may be a self-activating CD40 signaling protein that is capable of self-folding and transmitting a CD 40-mediated signal into APCs as if the signal had been contacted with another cell expressing CD40L (e.g., a CD4+ T cell). CD40 is a type 1 membrane protein with the N-terminus located outside the cell. CD40 is the primary switch (e.g., on dendritic cells), while CD40L (e.g., on CD4+ T cells, etc.) is a type 2 membrane protein with the C-terminus located outside the cell. Like many other members of the TNF family, CD40 requires trimerization to achieve signaling. Trimerization occurs through the interaction of CD40 with the trimerization domain of CD 40L. This activation requirement can be exploited to induce signaling by coupling CD40 to its own CD40L (with a trimerization domain) via a linker.

Thus, CD40/CD40L chimeras expressed in APCs must trimerize and achieve signaling without the need for another cell (typically a CD4+ T cell) to deliver CD 40L. Most preferably, the APC will also express or be exposed to the selected antigen and thus present a portion of the antigen on the MHC system. Such APCs enhance immune responses even in the absence of CD4+ T cells, which is significant in the infection by pathogens (e.g., HIV) that destroy or reduce CD4+ T cells. The immune response can be enhanced or down-regulated in a customized antigen-specific manner by co-presenting the chimeric protein with at least a portion of the antigen on the MHC. Chimeras can be trimerized for immune stimulation against specific antigens. Conversely, for immune downregulation, chimera trimerization can be reduced or inhibited.

Such constructs are particularly relevant to vaccines and other immunostimulatory compositions (particularly cancer vaccines), where the concept of trimerization is transferred to other TNF family members such as 4-1BB, OX40, etc., to activate cells through gene expression. Thus, the systems and methods described above are also suitable for uses other than APC (e.g., for NK cells and derivatives (e.g., NK-92, aNK, haNK, tank, etc.), T cells and derivatives (e.g., CAR-T, TCR-T, TIL-T, etc.), B cells, etc.).

For example, all CD40 variants are suitable for use herein. However, particularly suitable CD40 variants include human and other mammalian CD 40. Many such sequences are known (see, e.g., uniprot sequence databases), and all sequences are suitable for use herein. In certain non-limiting embodiments, the CD40 signal peptide is removed and replaced with an upstream portion comprising a linker and a CD40L portion. To activate the chimeric construct, CD40 will typically retain its intracellular activation domain. On the other hand, in cases where down-regulation is desired, CD40 will have an intracellular truncation lacking a (functional) activation domain.

Most typically, a particular CD40 will match an APC species (e.g., human CD40 for human APCs). Many modifications may be made to achieve the desired objectives. For example, the intracellular activation domain may be present in multiple copies, either partially deleted or completely deleted. In other examples, one or more amino acids may be added as tags for identification via immunohistochemistry. In still further examples, one or more amino acids may be exchanged (particularly at the N-terminus) to increase half-life. In a less preferred aspect, the CD40 transmembrane domain may be replaced with another transmembrane domain.

The sequence of CD40L may vary considerably. All CD40L variants are suitable for use herein. However, and as noted above, human and other mammalian CD40L are particularly suitable. Many such sequences are known (see, e.g., uniprot sequence databases), and all of these sequences are suitable for use herein. In certain non-limiting embodiments, CD40L will comprise its native signal peptide, however other signal peptides may also be included or substituted. CD40L should retain its trimerization domain to activate the chimeric construct. On the other hand, where down-regulation is desired, CD40L may have a truncated trimerization domain or some other sufficient steric hindrance to disrupt trimerization.

Most typically, CD40L will be selected to match the APC species (e.g., human CD40 for human APC, etc.). Many modifications may be made to achieve the desired objectives. For example, the trimerization domain may be optimized to increase affinity, or partially or completely deleted. In still further examples, one or more amino acids may be exchanged (particularly at the N-terminus) to increase half-life.

Suitable linkers typically provide sufficient mobility between the CD40 and CD40L moieties to allow all selective binding. Especially for activation of the chimeric molecule, the linker will be a polypeptide with between 4 and 60 amino acids, with low or no immunogenicity. Suitable linkers include GS-type linkers having between 8 and 50 or between 4 and 25, and most preferably between 15 and 17 amino acids. Many alternative linkers are known to exist (see, e.g., Adv Drug Deliv Rev. [ advanced Drug delivery review ] 201365 (10):1357-69), and all of these linkers are suitable for use herein.

Expression cassette

A recombinant expression cassette encoding the chimeric proteins described above may include a first nucleic acid segment encoding a portion of CD40L (the extracellular domain of CD40L and optionally a leader peptide coupled to the N-terminus of the extracellular domain of CD40L), a linker, and a portion of CD40 in a single reading frame, such that the portion of CD40L, linker, and portion of CD40 may be encoded in a single polypeptide. Exemplary chimeric constructs are shown in SEQ ID NOs 1-10. Where the leader peptide is to be coupled to the extracellular domain of CD40L, the segment of nucleic acid encoding the leader peptide may be placed in the same reading frame as the segment encoding the extracellular domain of CD40L (with or without a linker between them). The fusion protein may include an intervening sequence (e.g., a 2A sequence) or may be directly fused. The expression cassette includes a promoter (constitutive or inducible) to drive expression of the sequence encoding the chimeric protein. Since the chimeric protein has a transmembrane portion, the chimera will typically have a signal sequence (optionally cleavable) to direct the chimera to the cell surface.

Tumor associated antigens

Recombinant expression cassettes as described herein also typically include a second nucleic acid segment encoding a TAA (e.g., MUC1, CEA, short-tail mutein, RAS (e.g., mutated RAS (e.g., RAS having G12V, Q61R, and/or Q61L mutations, etc.)), tumor-specific antigen (e.g., PSA, PSMA, HER2), or tumor-and patient-specific neoantigen or neoepitope, which can be identified from patient omic data as used herein, "neoepitope" refers to a random mutation expressed in a tumor cell that constitutes a unique tumor-specific antigen. Unexpressed) mutation. The new sequence of tables may be defined to have a relatively short length (e.g., 8-12 me)r or a 14-20mer), wherein such extensions comprise one or more changes in amino acid sequence. Most typically, but not necessarily, the altered amino acid will be at or near the central amino acid position. For example, a typical new bit may have a₄-N-A₄Or A₃-N-A₅Or A₂-N-A₇Or A₅-N-A₃Or A₇-N-A₂Wherein a is a proprotein wild-type or normal (i.e., from the corresponding healthy tissue of the same patient) amino acid, and N is an altered amino acid (relative to wild-type or relative to matched normal). Thus, the new epitope sequence includes sequence extensions of relatively short length (e.g., 5-30mer, more typically 8-12mer or 14-20mer), where such extensions include one or more changes in amino acid sequence. Additional amino acids can be placed upstream or downstream of the altered amino acid, as desired, e.g., to allow additional antigen processing in various cellular compartments (e.g., proteasome, endosomes, lysosomes).

In some embodiments, the recombinant expression cassette can include sequences encoding one or more TAAs under separate promoters or in different reading frames such that the TAAs are expressed as separate molecules. In other embodiments, the recombinant expression cassette may comprise one or more sequences encoding TAAs a polyepitope. As used herein, "polyepitope" means a tandem array of two or more antigens expressed as a single polypeptide. Preferably, the two or more human disease-associated antigens are separated by a linker or spacer peptide. Any suitable length and order of peptide sequences for linkers or spacers may be used. However, the length of the linker is preferably between 3 and 30 amino acids, preferably between 5 and 20 amino acids, and more preferably between 5 and 15 amino acids. Glycine-rich sequences (e.g., gly-gly-ser-gly-gly, etc.) are preferred to provide multi-epitope flexibility between the two antigens. The second nucleic acid segment can further comprise a trafficking signal to direct a tumor-associated antigen, a tumor-specific antigen, a neoepitope, and/or a polyepitope to at least one of MHC-I and/or MHC-II complexes, more preferably at least to the MHC-II complex.

In some embodiments, the first and second nucleic acid segments are located in the same reading frame, preferably downstream of the same promoter, such that the chimeric protein and the tumor associated antigen can be expressed simultaneously. In other embodiments, an Internal Ribosome Entry Site (IRES) sequence separates the first and second nucleic acid segments such that translation of the first and second nucleic acid segments begins simultaneously. Alternatively, the sequence may also include an intervening sequence portion (e.g., a 2A sequence).

Additional molecules encoded by recombinant expression cassettes

In addition, the recombinant expression cassette may further comprise a third nucleic acid segment encoding one or more co-stimulatory molecules and/or cytokines to modulate an immune response in the tumor microenvironment. Suitable co-stimulatory molecules include B7.1(CD80), B7.2(CD86), CD30L, CD40, CD40L, CD48, CD70, CD112, CD155, ICOS-L, 4-1BB, GITR-L, LIGHT, TIM3, TIM4, ICAM-1, LFA3(CD58), and SLAM family members. Suitable cytokines include immunostimulatory cytokines (e.g., IL-2, IL-15, IL-17, IL-21, etc.) or down-regulating cytokines (e.g., IL-10, TGF-. beta.etc.) that attenuate the immune response. Alternatively or additionally, the nucleic acid may further comprise a sequence encoding at least one component of a SMAC (e.g., CD2, CD4, CD8, CD28, Lck, Fyn, LFA-1, CD43 and/or CD45 or respective binding counterparts thereof). In certain embodiments, the nucleic acid may further comprise a sequence encoding a STING pathway activator (e.g., a chimeric protein in which the transmembrane domain of LMP1 of EBV is fused to the signaling domain of IPS-1).

In a preferred embodiment, the cytokine is an IL-15 superagonist (IL-15N72D) and/or an IL-15 superagonist/IL-15 Ra Sushi-Fc fusion complex (e.g., ALT-803) coupled to at least one of IL-7, IL-15, IL-18, IL-21, and IL-22, or preferably both IL-7 and IL-21. Any suitable variation of IL-15 superagonists is contemplated. Exemplary and preferred embodiments of IL-15 superagonists are shown in FIGS. 10A-10F.

Expression vector

Most typically, the recombinant expression cassette is placed in an expression vector such that the nucleic acid segment encoding the peptide can persist through cell division. For example, the recombinant expression cassette is a DNA/RNA fragment, and a suitable DNA/RNA construct may be a linear or circular construct configured as an expression vector. Thus, in one embodiment, preferred expression vectors include viral vectors (e.g., non-replicating recombinant adenovirus genomes, optionally with deleted or non-functional E1 and/or E2b genes, etc.). The recombinant viruses so produced can then be used, alone or in combination, as therapeutic vaccines. Such vaccines are typically formulated as pharmaceutical compositions, e.g., sterile injectable compositions, with a viral titer of 10 per dosage unit⁶And 10¹³Between individual virus particles, and more typically 10⁹And 10¹²Between individual virus particles.

In still further embodiments, the expression vector may be a bacterial vector that can be expressed in genetically engineered bacteria that, when introduced into a human, express endotoxin at levels sufficiently low so as not to elicit an endotoxin response in human cells and/or to be insufficient to induce CD-14 mediated sepsis. Suitable bacteria include

BL21(DE3) electrocompetent cells. This strain is BL21 with the following genotype: F-ompT hsdSB (rB-mB-) gal dcm lon lambda (DE3[ lacI lacUV5-T7 gene 1ind1 sam7 nin5]) msbA148 Δ gutQ Δ kdsD Δ lpxL Δ lpxM Δ pagP Δ lpxP Δ eptA. Several specific deletion mutations (Δ gutQ Δ kdsD Δ lpxL Δ lpxM Δ pagP Δ lpxP Δ eptA) encode LPS-vs-lipid IV_AAnd an additional compensating mutation (msbA148) enables the cell to maintain viability in the presence of IVA. These mutations result in the deletion of the oligosaccharide chain from LPS, more specifically, two of the six acyl chains. Although electrocompetent BL21 bacteria are provided as examples, the genetically modified bacteria may also be chemically competent bacteria.

Alternatively or additionally, the expression vector may be a yeast vector that can be expressed in yeast. Preferred yeasts include Saccharomyces cerevisiae (e.g., GI-400 series recombinant immunotherapeutic yeast strains, etc.).

The recombinant nucleic acids described herein are not necessarily limited to viral, yeast, or bacterial expression vectors. Suitable vectors also include DNA vaccine vectors, linearized DNA, and mRNA, all of which can be transfected into suitable cells according to protocols well known in the art.

Viral vaccine formulations and applications

The recombinant nucleic acids (or recombinant expression cassettes) and/or recombinant viruses carrying the recombinant nucleic acids can be used to induce or generate antigen presenting cells (e.g., dendritic cells) in vivo or ex vivo. The resulting chimeric proteins and TAAs can enhance the anti-tumor immune response against cells expressing TAAs. One or more recombinant viruses comprising one or more nucleic acid segments encoding a chimeric protein and/or one or more tumor-associated antigens, cytokines, and/or co-stimulatory molecules can be administered to the patient's APCs in vivo. Such infected APCs express one or more TAAs, cytokines, and/or co-stimulatory molecules to stimulate an immune response against the tumor cells.

For example, a genetically modified virus carrying a recombinant nucleic acid encoding a chimeric protein and/or one or more TAAs can be formulated in any pharmaceutically acceptable carrier (e.g., preferably as a sterile injectable composition, etc.) to form a pharmaceutical composition. The sterile composition may be administered by any suitable method. In some embodiments, when a cytokine (e.g., ALT-805) is to be expressed in the same cell, the recombinant nucleic acid further comprises a nucleic acid encoding the cytokine. Additionally or alternatively, another recombinant virus (or bacteria or yeast) comprising a recombinant nucleic acid encoding a cytokine may be co-administered. When it is desired that two or more types of recombinant viruses infect the same antigen presenting cell, the two or more types of recombinant viruses may be formulated into a single pharmaceutical composition. However, two or more types of recombinant viruses can also be formulated into two separate and distinct pharmaceutical compositions and administered to a patient simultaneously or substantially simultaneously (e.g., within one hour, within 2 hours, etc.).

Where the pharmaceutical composition comprises a recombinant virus, the titer should be 10 per dosage unit⁴And 10¹²Between individual virus particles. However, alternative formulations are also suitable for use herein, as well as all known routes and modes of administration. Where the pharmaceutical composition comprises a recombinant bacterium, the titer should be 10 per dosage unit²And 10³Between individual bacteria, 10³And 10⁴Between individual bacteria, or 10⁴And 10⁵Between individual bacteria. In the case where the pharmaceutical composition comprises recombinant yeast, the titer should be 10 per dosage unit²And 10³Between individual yeasts, 10³And 10⁴Between individual yeasts, or 10⁴And 10⁵Between the individual yeasts.

As used herein, "applying" a viral, bacterial, or yeast formulation refers to both direct and indirect application. Direct application of the formulation is typically performed by a health care professional (e.g., physician, nurse, etc.). Indirect application includes the step of providing a formulation to or making available to a health care professional a formulation for direct application (e.g., via injection, infusion, oral delivery, topical delivery, etc.).

In some embodiments, the viral, bacterial or yeast formulation is injected systemically, including subcutaneous (subdutaneous), subcutaneous (subdermal), or intravenous injection. In other embodiments, the formulation may be injected into a tumor in cases where systemic injection may not be effective (e.g., for brain tumors, etc.).

The dose and/or schedule of administration may vary depending on: the type of virus, bacteria or yeast, the type and prognosis of the disease (e.g., tumor type, size, location), and the health condition of the patient (e.g., including age, sex, etc.). Although the dosage and schedule may vary, they may be selected and adjusted so that the formulation has little significant toxic effect on normal host cells, but is sufficient to elicit an immune response. Thus, in a preferred embodiment, the optimal administration may be determined based on a predetermined threshold. For example, the predetermined threshold can be a predetermined local or systemic concentration of a particular type of cytokine (e.g., IFN- γ, TNF- β, IL-2, IL-4, IL-10, etc.). Typically the dosage, route and schedule are adjusted so that at least local or systemic expression of immune response specific cytokines is at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, more.

For example, where the pharmaceutical composition comprises a recombinant virus, the dose is at least 10⁶Individual virus particles/day, or at least 10⁸Individual virus particles/day, or at least 10¹⁰Individual virus particles/day, or at least 10¹¹Individual virus particles per day. In some embodiments, a single dose of the viral formulation may be administered at least once daily or twice daily (half dose per administration) for at least one day, at least 3 days, at least one week, at least 2 weeks, or at least one month. In other embodiments, the dosage of the viral formulation may be gradually increased during the schedule, or gradually decreased during the schedule. In still other embodiments, several series of formulations may be applied, with the application of each series separated by an interval time (e.g., one series application for 3 consecutive days each, and 7 days apart and one series application for 3 consecutive days each, etc.).

In some embodiments, the formulation may be applied in two or more stages: for example, a primary application and a booster application. The initial dose may be higher (e.g., at least 20%, preferably at least 40%, more preferably at least 60%, etc.) than the subsequent booster dose. Alternatively, the initial dose may be lower than the subsequent booster dose. In addition, where there are multiple boosts, each boost can have a different dose (e.g., an increased dose, a decreased dose, etc.).

Cell-based compositions and administration

The patient's own APCs can be isolated from the blood and transfected with recombinant nucleic acids encoding chimeric proteins and/or TAAs. Isolated patient APCs may also be infected with recombinant viruses, bacteria, or yeast containing recombinant nucleic acids. In some embodiments, MHC-matched heterologous APCs can be used with or in place of patient's own APCs. For example, patient dendritic cells (allogeneic dendritic cells) can be isolated and further expanded ex vivo with TNF- α, granulocyte-macrophage colony stimulating factor (GM-CSF), or Interleukin (IL) -4. These Dendritic Cells (DCs) can then be further transfected with recombinant nucleic acids or infected with a viral vaccine comprising recombinant nucleic acids. Optionally, after transfection and/or infection, infected and/or transfected DCs may be further expanded ex vivo to increase the population of DCs to be administered.

Transfected/infected DCs can be formulated in any pharmaceutically acceptable carrier (e.g., as a sterile injectable composition) with cell titers of at least 10 per dosage unit³Individual cells/mL, preferably at least 10⁵Individual cells/mL, more preferably at least 10⁶Individual cells/mL, and at least 1mL, preferably at least 5mL, more preferably and at least 20 mL. Alternative formulations are also suitable for use herein, as well as all known routes and modes of administration.

Adjuvants to be co-administered

The virus, bacterium or yeast having a recombinant nucleic acid encoding a chimeric protein and/or one or more TAAs may be co-administered with one or more adjuvants and/or additional molecules to enhance the effect. For example, expression of a viral payload (recombinant nucleic acid or cassette) and/or viral infection efficiency can be greatly increased by co-administration or co-exposure of non-host cells as adjuvants. Suitable non-host cells may include cells belonging to a species other than the host cell species (e.g., human for a patient), or cells belonging to the host species but exhibiting one or more stress or danger signals (e.g., cells exposed to chemotherapeutic agents, radiation, etc. to trigger NKG2DL expression, stress markers, pro-apoptotic markers, etc.). Most typically, however, suitable non-host cells will be pathogenic or otherwise bacterial and/or yeast.

For example, suitable bacteria include bacteria modified to have reduced expression of LPS, which would otherwise trigger an immune response and elicit an endotoxin response. An exemplary bacterial strain having a modified lipopolysaccharide includes

BL21(DE3) electrocompetent cells. Although electrocompetent BL21 bacteria are provided as examples, suitable genetically modified bacteria may also be chemically competent bacteria.

Alternatively, inactive or attenuated mycobacterium bovis (e.g., bacillus calmette-guerin) may be an adjuvant. In addition, the patient's own endosymbiotic bacteria can act as non-host cells. As used herein, a patient's "endosymbiotic bacteria" refers to bacteria that are present in the patient without eliciting any substantial immune response. Thus, the patient's endosymbiont bacteria is the patient's normal flora. Endosymbiotic bacteria may include the genera Escherichia coli or Streptococcus, which are commonly found in the human intestine or stomach. Endosymbiotic bacteria can be obtained from patient biopsy samples from the intestine, stomach, oral mucosa, conjunctiva, or from stool samples. The patient's endosymbiotic bacteria can then be cultured and transfected with nucleotides encoding one or more human disease-associated antigens. Bacterial non-host cells may also include pathogenic cells including bordetella pertussis and/or mycobacterium bovis. Most typically, but not necessarily, the bacterial non-host cells will be killed prior to exposure to the host cells.

Many yeast strains are suitable for use herein. Typical non-pathogenic yeasts include Saccharomyces cerevisiae, Saccharomyces boulardii (S.boulardii), Pichia pastoris (Pichia pastoris), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Candida stellata (Candida stellata), and the like. Such yeast strains may be further genetically modified to reduce one or more undesirable traits, and/or to express recombinant proteins that further increase viral infectivity and/or expression. Suitable yeast strains are generally commercially available and can be modified via known protocols.

Without being bound by theory, one or more non-host cell components may serve as a danger or destruction signal, particularly where the host cell is an immune competent cell. Thus, not only non-host cells, but also one or more immunostimulatory portions thereof, may be used. Suitable moieties include PAMP receptor ligands, DAMP receptor ligands, TLR ligands, CpG, ssDNA, and thapsigargin.

The exact ratio of non-host cells to host cells can vary considerably depending on the type of host cell, the type of non-host cell (or components thereof), and the virus (or DNA/RNA). However, typically the ratio of host cell to non-host cell is from 1:1 to about 1:100, or from 1:10 to about 1:1,000, or from 1:50 to about 1:5,000, or from 1:100 to about 1:10,000, especially where the immune competent cell is a host cell and the bacterial cell is a non-host cell. Similarly, suitable ratios of host cells to non-host cells include 100:1 to about 10:1, or 1:1 to about 1:10, or 1:50 to about 1:5,00, or 1:100 to about 1:1,000, particularly where the host cells are immune competent cells and the non-host cells are yeast.

In the presence of non-host cells, the exposure of the host cells to the recombinant virus (or DNA/RNA) may be quite different. However, typically the exposure time is between a few minutes and a few hours, or a few hours to a few days. For example, where exposure is performed in vitro, the exposure time may be between 10 minutes and 2 hours, or between 30 minutes and 4 hours, or between 60 minutes and 6 hours, or between 2 hours and 8 hours, or between 6 hours and 12 hours, or between 12 hours and 24 hours, or between 24 hours and 48 hours, or even longer. On the other hand, when the exposure is performed in vivo (e.g., via a vaccine formulation), the exposure time may be between 60 minutes and 6 hours, or between 6 hours and 12 hours, or between 12 hours and 24 hours, or between 24 hours and 48, or even longer. In such vaccination protocols, host cells, non-host cells, and recombinant viruses (or DNA or RNA) may be co-administered in the same formulation.

A viral, bacterial, or yeast formulation having a recombinant nucleic acid encoding a chimeric protein and/or one or more TAAs can be co-administered with one or more cytokines and/or checkpoint inhibitors. Any cytokine that modulates an immune response (e.g., increases or decreases T cell activity, etc.) will work. Most preferably, the cytokine is an IL-7, ILAt least one of-15, IL-18, IL-21 and IL-22, or preferably both IL-7 and IL-21, coupled IL-15 superagonists (IL-15N72D), and/or IL-15 superagonists/IL-15 Ra Sushi-Fc fusion complex (e.g., ALT-803). Exemplary cytokines are shown in FIGS. 10A-10F. Exemplary checkpoint inhibitors include those directed against CTLA-4 (particularly against CD 8)⁺Cells), PD-1 (especially against CD 4)⁺Cells), TIM1 receptor, 2B4, and CD160 antibodies or binding molecules. Ipilimumab and nivolumab are suitable checkpoint inhibitors.

Without wishing to be bound by theory, co-administration of the recombinant virus and transfected/infected APCs to a patient will activate T cells against tumor cells that express TAAs in the tumor microenvironment by increasing the number of pre-activated APCs (e.g., DCs) that present the TAAs and by exposing such APCs to helper T (th) cells or other immune cells. Th cells interacting with such APCs may further activate a signaling cascade to generate more memory T cells and elicit an immune response against tumor cells.

Examples of the invention

The crystal structures of CD40, CD40L, CD40/CD40L complexes were used to determine the range of linker lengths that can tether CD40 and CD40L together while maintaining functionality. For this purpose, five linkers of different lengths were modeled and expressed recombinantly. Several fusion proteins were tested.

FIG. 1 depicts an exemplary 16-mer linker model carrying a fusion protein. The left panel shows a predicted side view of the chimeric protein monomer. The middle panel depicts a side view of the prediction of the trimer. The right panel depicts a top view of the prediction of the trimer. As can be seen, the linker provides sufficient spatial mobility to allow CD40L to bind to CD40 and allow trimerization.

To determine whether these constructs would also stimulate immune competent cells, KG-1 cells (myeloid cell line) were transfected with constructs with different linker lengths. These cells were transfected at about 30% -50%. KG-1 cells were transfected by electroporation using a BioRad Gene Pulser II at 3 pulses (200 ohms, 25. mu.f, 0.26kV) and then cultured in growth Medium (Iscove's Modified Dulbecco's Medium) supplemented with 20% fetal bovine serum for 16 hours. Transfected cells were washed to remove residual cytokines that may have been produced by the electroporation process and cultured in fresh medium in 96-well tissue culture plates at 20,000 cells per well. The cells were cultured for another 24 hours, and the supernatant was harvested. Cytokine levels in the supernatants were determined using a flow microbead array method specific for human IL-1 β, MCP-1 and IL-8 according to the manufacturer's recommended protocol; however, only IL-8 showed any change. FIG. 2 shows IL-8 from human cells transiently transfected with CD 40L-linker-CD 40 constructs with varying linker lengths. Linker lengths of about 16 amino acids were found to be most effective.

Mouse CD40L/CD40 fusion protein: to determine whether the concept of the self-ligating CD40/CD40L fusion construct could be extended to other species, a parallel set of mouse-type constructs encoding these proteins was produced and tested for activity in the mouse B16F10 melanoma cell line. B16F10 cells were transfected with the mouse CD40/CD40L fusion protein construct using Lipofectamine 2000 according to the manufacturer's recommended protocol. The cells were left for 18 hours, washed to remove residual cytokines and cultured in fresh growth medium (DMEM supplemented with 10% FBS) in 96 well tissue culture plates for an additional 24 hours at 50,000 cells per well. After incubation, the supernatants were harvested and the levels of IL-1 β, MCP-1 and KC in mice were determined using the flow bead array method according to the manufacturer's recommended protocol. Figure 3 shows that similar results were obtained in this parallel system, suggesting that the system may be extended to other CD40 sequences and even other TNF family members. Some constructs triggered a large number of activities in transfected cells (both KC and MCP-1), indicating that linker lengths of 14 or 16 amino acids were most effective. The 18 amino acid linker did not elicit a response.

IL-8 secretion was determined by transfecting dendritic cell-like (KG-1) and 293T derivative (EC7) cells with the chimeric constructs using essentially the same protocol as described above. Figure 4 shows that both cell lines have significant IL-8 secretion in all variants tested. To further test whether these constructs could be manipulated across species boundaries, mouse melanoma cells (B16F10) were transfected with both human and mouse constructs, and the secretion of KC and MCP-1 was determined. FIG. 5 shows the secretion of KC and MCP-1 even when the chimeric constructs are not from the same species.

Human (293T) and murine (B16F10) cells were transfected and labeled with monoclonal or polyclonal antibodies 24 hours later. FIGS. 6 and 7 show that for all constructs, the CD40/CD40L construct was expressed on the surface of both cell lines.

The chimeric constructs were tested for functionality against 293T transfected with CD40, followed by sCD40L stimulation. The results are shown in FIG. 8. Notably, the chimeric construct induced more IL-8 secretion than the soluble CD40 ligand. Finally, chimeric constructs were made using mouse and human sequence elements for the CD40 domain of the fusion protein. Thus, Intracellular (IC), transmembrane^TMOr Extracellular (EC) domain, at least some of the fusion proteins are also chimeric. Notably, figure 9 shows that chimeric constructs in human cells using human EC elicit significantly higher IL-8 secretion even with murine IC and TM segments. Similarly, human EC was also superior in murine cells.

In a preferred embodiment, the CD40/CD40L protein construct is illustrated in the accompanying sequence listing. No. 1 is an illustrative example of a human CD40/CD40L construct with a 12mer linker. SEQ ID No. 2 is an illustrative example of a human CD40/CD40L construct with a14 mer linker. No. 3 is an illustrative example of a human CD40/CD40L construct with a 16mer linker. No. 4 is an illustrative example of a human CD40/CD40L construct with an 18mer linker. SEQ ID No. 5 is an illustrative example of a human CD40/CD40L construct with a 20mer linker. NO. 6 is an illustrative example of a mouse CD40/CD40L construct with a 12mer linker. NO. 7 is an illustrative example of a mouse CD40/CD40L construct with a14 mer linker. NO. 8 is an illustrative example of a mouse CD40/CD40L construct with a 16mer linker. SEQ ID No. 9 is an illustrative example of a mouse CD40/CD40L construct with an 18mer linker. No. 10 is an illustrative example of a mouse CD40/CD40L construct with a 20mer linker. Other constructs of 4-1BBL/4-1BB and Ox40L/Ox40 may be based on publicly available Uniprot sequences in a manner substantially as described above for CD40L/CD 40.

In some preferred embodiments, the genetically engineered activated dendritic cells can be prepared by infecting a tumor cell with a recombinant nucleic acid having a first and a second nucleic acid segment; wherein the first nucleic acid segment encodes a chimeric protein having an extracellular portion of CD40 coupled to CD40L through a flexible linker; and wherein the second nucleic acid segment encodes a tumor associated antigen. The genetically engineered activated dendritic cells can further comprise a recombinant nucleic acid encoding an antibody secreting portion to affect the tumor microenvironment. The antibody secreting portion may comprise one or more of: PD1, CTLA4 and TGFb trap and IL 8.

In some embodiments, genetically engineered activated DCs can be used to treat tumors. The method comprises administering to a patient a composition comprising genetically engineered activated DCs as discussed above. Administration of genetically engineered activated DCs can be performed as follows: small scale (bladder cancer, brain cancer), or local (skin tumor), or interventional injection into tissue (liver cancer, breast cancer, pancreatic cancer), or inhalation (lung cancer, or brain cancer), or intrathecal. In some embodiments, the tumor killing properties of the engineered cells can be further enhanced via CD46 to target both the CAR and CD 46. Furthermore, the methods and engineered cells disclosed herein can be used as disclosed in: do et al (2018) int.J.mol.Sci. [ International journal of molecular science ]19:2694 and Zhai et al (2102) Gene Ther. [ Gene therapy ]19(11): 1065-74.

Neoepitope-based immunotherapy can be improved by using adjuvants that are co-expressed or co-presented with immunogenic peptides, preferably patient and tumor specific neoepitopes. Most typically, expressed patient-and tumor-specific neo-epitopes target processing and/or specific cell surface presentation or even secretion. When desired, checkpoint inhibition, immune stimulation via cytokines, and/or inhibitors of bone marrow-derived suppressor cells (MDCS), T regulatory cells (tregs), or M2 macrophages may still be used to further enhance neoepitope-based therapies.

By way of non-limiting example, such therapeutic entities will be expressed in vivo from recombinant nucleic acids, and particularly suitable recombinant nucleic acids include plasmids and viral nucleic acids. In the case of viral nucleic acids, it is particularly preferred to deliver the nucleic acids via viral infection of patient cells.

The compositions and methods presented herein will deliver an adjuvant in the presence and/or expression of one or more neoepitopes. Indeed, such treatments may be advantageously tailored to achieve one or more specific immune responses (including CD 4)⁺Biased immune response, CD8⁺Biased immune responses, antibody biased immune responses, and/or stimulated immune responses (e.g., reduction of checkpoint inhibition and/or activation of immune competent cells by use of cytokines)), all of which can benefit from the presence of an adjuvant. In the absence of expression of an adjuvant (e.g., the adjuvant is ATP or an ATP analog), it is preferred that the adjuvant is injected into the tumor such that the vaccine composition and adjuvant are present at the same time (e.g., the vaccine composition and adjuvant are present at the same time in a measurable amount).

All known adjuvants are suitable for use herein. Suitable exemplary adjuvants include various inorganic compounds, such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, mineral oil, and especially paraffin oil. Other suitable adjuvants include small molecule compounds such as squalene, as well as various bacterial products such as the killed bacteria bordetella pertussis, mycobacterium bovis toxoid, and the like. Adjuvants may also be formed from one or more emulsified neo-antigens to produce a complex composition, such as Freund's complete adjuvant or Freund's incomplete adjuvant.

Particularly suitable adjuvants include various DAMPs (damage-associated molecular pattern proteins). DAMPs are known to trigger inflammatory, innate and adaptive immune responses, and tissue healing following injury. Particularly preferred DAMPs include calreticulin or a portion thereof having adjuvant activity, and HMGB1 or a portion thereof having adjuvant activity. Still other DAMPs include the S100 protein and various cytokines, and in particular IL-1, IL-2, and IL-12.

HMGB1 is a damage-associated molecular pattern (DAMP) protein that is normally released inside cells but after cell death to allow immunological differentiation between dangerous and harmless antigens. Cells experiencing severe stress secrete HMGB 1. Extracellular HMGB1 triggers inflammation and an adaptive immune response. HMGB1 has also been reported to enhance the immunogenicity of muteins (neoantigens or neo-epitopes) in tumors, thereby promoting anti-tumor responses and immunological memory (see, e.g., Immunol Rev. [ immunological review ] (2017)280(1): 74-82). For example, HMGB1 was reported to induce dendritic cell maturation and T helper 1 cell responses.

Specific fragments of HMGB1 were reported to activate dendritic cells (see US 9,539,321). Peptides comprising the sequence SAFFLFCSE have an immunostimulatory effect in vitro, and such sequences can be attached to nanoparticles or microparticles. HMGB1 also promotes the maturation of antigen presenting cells (US 2004/0242481). As described in US 2011/0236406, portions of HMGB1 were used in fusion proteins to activate T cells.

Cancer therapy can induce a stress response in the ER, translocating calreticulin to the outer leaflet of the plasma membrane before the morphology of apoptosis occurs. This surface exposed calreticulin serves as a powerful mobilization signal for the immune system. Therefore, the externally added calreticulin is an immunopotentiator in the context of photodynamic therapy of tumors (Front immune [ immune Front ] (2015)5(15): 1-7).

HMGB1 and calreticulin adjuvants require the formulation of a mixture of vaccine compounds and adjuvants in a more traditional manner, or the systemic administration of adjuvants outside the antigenic range of calreticulin. However, such methods are generally not suitable for cancer therapy, especially where the cancer antigen is a recombinant antigen.

Advantageously, the polypeptide or protein adjuvant may be administered in the direct context of the neo-epitope (or TAA or tumor-specific antigen) by co-expression of the polypeptide or protein adjuvant together with the neo-epitope, as described herein. Such co-expression may be carried out in living cells, in particular in APCs of a patient diagnosed with a tumor, or in a yeast or bacterial vaccine composition administered to the patient.

For example, as described in more detail below, recombinant nucleic acids can be constructed that include one or more expression cassettes for expression of the neoepitope (preferably in a manner that directs the neoepitope to MHC-I and/or MHC-II presentation), and further include expression cassettes encoding one or more polypeptides or protein adjuvants. The polypeptide or protein adjuvant may be expressed as a membrane-bound protein or a soluble secreted protein. The recombinant nucleic acid can further include a sequence encoding at least one of a co-stimulatory molecule, an immunostimulatory cytokine, and a protein that interferes with or down-regulates checkpoint inhibition. Suitable co-stimulatory molecules include OX40L, 4-1BBL, CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1 and LFA3, and suitable immunostimulatory cytokines include IL-2, IL-12, IL-15 superagonists (ALT803), IL-21, IPS1 and LMP 1. Preferred proteins that interfere with checkpoint inhibition include antibodies or antagonists to CTLA-4, PD-1, TIM1 receptor, 2B4 or CD 160.

Additionally or alternatively, non-protein stress signals can be delivered to the tumor as part of immunotherapy via systemic administration or intratumoral administration. For example, non-protein adjuvants include various purine metabolites, particularly ATP and ATP analogs (e.g., non-hydrolyzable α, β -methylene-ATP (α β -ATP)). Extracellular ATP serves as a danger signal alerting the immune system to tissue damage and triggers DC activation.

Cancer immunotherapy may use recombinant adenoviruses. Such adenoviruses may carry cancer epitopes as payload, and at least one polypeptide or protein adjuvant, and optionally additional functional elements as discussed below. Cancer epitopes are typically tumor and patient specific neoepitopes filtered according to one or more criteria (also described below).

Neo-epitope identification can begin with a variety of biological materials, including fresh biopsies, frozen or otherwise preserved tissue or cell samples, circulating tumor cells, exosomes, a variety of bodily fluids (especially blood), and the like. Suitable omic analysis methods include nucleic acid sequencing, and in particular NGS methods that operate on DNA (e.g., Illumina sequencing, ion torrent sequencing, 454 pyrosequencing, nanopore sequencing, etc.); RNA sequencing (e.g., RNAseq, reverse transcription based sequencing, etc.); and in some cases protein sequencing or mass spectrometry based sequencing (e.g., SRM, MRM, CRM, etc.).

For nucleic acid-based sequencing, high-throughput genomic sequencing of tumor tissue allows rapid identification of neoepitopes. However, normally occurring inter-patient variation (e.g., due to SNPs, short indels (indels), different numbers of repeats, etc.) and heterozygosity will result in a relatively large number of potentially false positive neoepitopes (i.e., neoepitopes also found on healthy tissue of the same patient) when comparing sequence information to a standard reference sequence. Notably, this inaccuracy can be eliminated in the case of comparing a patient's tumor sample with a matched normative (i.e., non-tumor) sample of the same patient.

DNA analysis can be performed by whole genome sequencing and/or exome sequencing (usually at least 10x, more typically at least 20x by depth of coverage) of both the tumor sample and the matched normal sample. Alternatively, DNA data can also be provided from established sequence records (e.g., SAM, BAM, FASTA, FASTQ, or VCF files) from previous sequence determinations from the same patient. Suitable data sets include unprocessed or processed data sets, and exemplary preferred data sets include those having a BAM format, a SAM format, a GAR format, a FASTQ format, or a FASTA format, as well as BAMBAM, SAMBAM, and VCF data sets. However, as described in US 2012/0059670 and US 2012/0066001, BAM format or bambambam diff objects are particularly suitable. The data set reflects tumor samples and matched normative samples of the same patient. Thus, genetic germline changes (e.g., silent mutations, SNPs, etc.) that do not cause tumors can be excluded. The tumor sample may be from the original tumor, from the tumor at the beginning of the treatment, from a recurrent tumor and/or metastatic site, etc. In most cases, the patient's matched normal sample is blood or non-diseased tissue from the same tissue type as the tumor.

Likewise, sequence data can be analyzed in a number of ways. However, in the most preferred method, as in US 2012/0059670 and US 2012/0066001, in silico analysis is performed using BAM files and BAM servers through position-guided simultaneous alignment of tumor and normal samples. Such an analysis advantageously reduces false positive neo-epitopes and significantly reduces the need for memory and computing resources.

Any language referring to "computer" should be read to include any suitable combination of computing devices, including servers, interfaces, systems, databases, agents, terminals, engines, controllers, or other types of computing devices operating alone or in combination. The computing device includes a processor configured to execute software instructions stored on a tangible, non-transitory computer-readable storage medium (e.g., hard disk drive, solid state drive, RAM, flash memory, ROM, etc.). The software instructions preferably configure the computing device to provide roles, responsibilities, or other functions as discussed below with respect to the disclosed apparatus. Furthermore, the disclosed techniques may be embodied as a computer program product that includes a non-transitory computer-readable medium storing software instructions that cause a processor to perform the disclosed steps associated with the implementation of computer-based algorithms, processes, methods, or other instructions. In a particularly preferred embodiment, the various servers, systems, databases, or interfaces exchange data using standardized protocols or algorithms, possibly based on HTTP, HTTPs, AES, public-private key exchanges, web services APIs, known financial transaction protocols, or other electronic information exchange methods. Data exchange between devices may be through a packet-switched network, the internet, a LAN, a WAN, a VPN, or other type of packet-switched network; a circuit-switched network; a cell switching network; or other type of network.

A collection of patient-and cancer-specific in silico sequences can be established that encode new epitopes having a predetermined length of, for example, between 5 and 25 amino acids, and that include at least one altered amino acid. Such a collection will typically include at least two, at least three, at least four, at least five, or at least six members for each altered amino acid, where the positions of the altered amino acids are different. Such a collection advantageously increases the potential candidate molecules for immunotherapy and may then be used for further filtering (e.g., by subcellular localization, transcription/expression levels, MHC-I and/or II affinities, etc.), as described in more detail below.

For example, using synchronized location-guided analysis of tumor and matched normal sequence data, various cancer neoepitopes have been identified from a variety of cancers and patients, including the following cancer types: BLCA, BRCA, CESC, COAD, DLBC, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, OV, PRAD, READ, SARC, SKCM, STAD, THCA, and UCEC. Exemplary new epitopes for these cancers can be found in international application PCT/US16/29244, which is incorporated herein by reference.

Depending on the type and stage of the cancer, and the immune status of the patient, not all of the identified new epitopes necessarily result in a therapeutically equivalent response by the patient. Indeed, only a fraction of neoepitopes will generate an immune response. To increase the likelihood of a therapeutically desirable response, the initially identified new epitopes may be further filtered. For the purposes of the methods presented herein, downstream analysis need not take into account silent mutations. However, a preferred mutation analysis will provide information on the impact of the mutation (e.g., nonsense, missense, etc.) in addition to the particular type of mutation (e.g., deletion, insertion, transversion, transition, translocation), and may thus serve as a first content filter through which silent mutations are eliminated. For example, a new epitope may be selected for further consideration, where the mutation is a frameshift, nonsense, and/or missense mutation.

In a further filtering approach, the neo-epitopes can also be analyzed in detail for subcellular localization parameters. For example, if a neo-epitope is identified as having a membrane-associated position (e.g., located outside of the cell membrane of a cell) and/or if in silico structure calculations confirm that the neo-epitope may be exposed to solvents, or exhibit a structurally stable epitope (e.g., J Exp Med [ journal of experimental medicine ]2014), etc., the neo-epitope sequence may be selected for further consideration.

The neoepitope is particularly suitable for use where omics or other analysis reveals actual expression of the neoepitope. The expression and expression level of the novel epitope can be identified in all ways known in the art. Preferred methods include quantitative RNA (hnRNA or mRNA) analysis and/or quantitative proteomic analysis. Most typically, the threshold level comprising the neo-epitope will be an expression level that is at least 20%, at least 30%, at least 40%, or at least 50% of the expression level of the corresponding matching normal sequence, thus ensuring that the (neo) epitope is at least potentially 'visible' to the immune system. Therefore, it is generally preferred that omics analysis also include analysis of gene expression (transcriptomics analysis), thereby facilitating the identification of the expression level of genes having mutations.

Many transcriptomics analysis methods are known in the art, and all known methods are suitable for use herein. For example, mRNA and primary transcript (hnRNA) and RNA sequence information can be derived from reverse transcribed polyadenylic acid⁺-RNA(polyA⁺-RNA), the reverse transcription of polyadenylic acid⁺RNA was in turn obtained from tumor samples and matched normal (healthy) samples of the same patients. Also, despite polyadenylic acid⁺RNA is typically preferred as representative of the transcriptome, but other forms of RNA (hn-RNA, non-polyadenylated RNA, siRNA, miRNA, etc.) are also suitable. Preferred methods include quantitative RNA (hnRNA or mRNA) analysis and/or quantitative proteomic analysis, including RNAseq in particular. In other aspects, RNA quantification and sequencing is performed using RNAseq, qPCR and/or rtPCR based methods, although a variety of alternative methods (e.g., solid phase hybridization based methods) are also suitable. Transcriptomic analysis (alone or in combination with genomic analysis) may be suitable for identifying and quantifying genes with cancer-and patient-specific mutations.

Similarly, proteomic analysis can be performed in many ways to determine the actual translation of the neoepitope's RNA, and all known proteomic analyses are suitable. Preferred proteomics methods include antibody-based methods and mass spectrometry methods. Proteomic analysis can provide qualitative or quantitative information about the protein itself, but can also include protein activity data where the protein has catalytic or other functional activity. See, for example, US 7,473,532, which is incorporated herein by reference. Other suitable methods of identifying and even quantifying protein expression include various mass spectrometric analyses (e.g., Selective Response Monitoring (SRM), Multiple Response Monitoring (MRM), and Continuous Response Monitoring (CRM)). The above methods will provide patient and tumor specific neo-epitopes that can be further filtered by the subcellular location (e.g., membrane location), the strength of expression (e.g., over-expressed as compared to a matched normal control for the same patient), etc. of the protein comprising the neo-epitope.

The neoepitope can be compared to a database containing known human sequences (e.g., sequences of a patient or a collection of patients) to avoid using the same sequences as human. Moreover, filtering may also include removing new epitope sequences due to SNPs in the patient, where these SNPs are present in both the tumor and the matching normal sequence. For example, dbSNP (single nucleotide polymorphism database) is a free public archive of genetic variation within and between different species developed and hosted by the National Center for Biotechnology Information (NCBI) in collaboration with the national institute of human genome (NHGRI). Although the name of the database implies only a collection of one type of polymorphism (single nucleotide polymorphisms (SNPs)), in practice it contains a relatively broad range of molecular variations: (1) SNP; (2) deficiency and insertion polymorphism (indels/DIPs); (3) microsatellite markers or Short Tandem Repeats (STRs); (4) a polynucleotide polymorphism (MNP); (5) a heterozygous sequence; and (6) the named variants. dbSNP receives apparently neutral polymorphisms corresponding to polymorphisms of known phenotype and regions with no variation. Using such a database and other filtering options as described above, patient and tumor specific neo-epitopes can be filtered to remove those known sequences, resulting in a sequence set with multiple new epitope sequences with significantly reduced false positives.

Once the neoepitope is sufficiently filtered (e.g., by tumor versus normal control, and/or expression level, and/or subcellular location, and/or patient-specific HLA matching, and/or known variants), further filtering steps can take into account the gene type affected by the neoepitope. For example, suitable gene types include cancer driver genes, genes associated with the regulation of cell division, genes associated with apoptosis, and genes associated with signal transduction. However, in particularly preferred aspects, cancer driver genes are particularly preferred (which can persist through the functionalization of a variety of gene types, including receptor genes, signal transduction genes, transcriptional regulatory genes, and the like). Suitable gene types may also be the known passenger genes (passanger genes) and genes involved in metabolism.

Various methods and predictive algorithms are known in the art to determine whether a gene is a cancer driver gene. For example, suitable algorithms include Mutsig CV (Nature [ Nature ]2014,505(7484):495-501), Activedriver (Mol Syst Biol [ molecular systems biology ]2013,9:637), MuSiC (Genome Res [ Genome research ]2012,22(8):1589-1598), Oncorivecclusion (Bioinformatics [ Bioinformatics ]2013,29(18): 2238-. Alternatively or additionally, the identification of cancer driver genes may also employ multiple sources to obtain known cancer driver genes and their association with a particular cancer. For example, the Intogen catalog of driver mutations (2016.5; URL: www.intogen.org) contains the results of a driver analysis by a Cancer Genome Interpreter (Cancer Genome Interpreter) of 6,792 exons in a pan-Cancer cohort of 28 tumor types.

However, despite the filtering, not all neo-epitopes are visible to the immune system, as these neo-epitopes need to be processed in the presence of a larger background (e.g., within multiple epitopes) and presented on the MHC complex of the patient. Only a portion of all neoepitopes will have sufficient affinity for presentation. Thus, where neoepitopes are properly processed by, bound to, and presented by MHC complexes, these neoepitopes will be more likely to be effective. Treatment success will increase with an increase in the number of neoepitopes that can be presented via MHC complexes, where such neoepitopes have minimal affinity for the patient's HLA type. Efficient binding and presentation is the combined function of the sequence of the neoepitope and the particular HLA type of the patient. Thus, HLA type determination of patient tissue is often required. Most typically, the HLA class determination comprises at least three MHC-I subtypes (e.g., HLA-A, HLA-B, HLA-C) and at least three MHC-II subtypes (e.g., HLA-DP, HLA-DQ, HLA-DR), preferably wherein each subtype is determined to be at least 2-bit, at least 4-bit, at least 6-bit, or at least 8-bit deep.

Once the HLA type of the patient is determined, structural solutions for the HLA type can be calculated and/or obtained from the database and then used in a docking model in a computer to determine the (typically filtered) binding affinity of the neo-epitope to the HLA structural solution. Suitable systems for determining binding affinity include the NetMHC platform (see, e.g., Nucleic Acids Res. [ Nucleic Acids research ] 2008. 7.1; 36(Web Server issue): W509-W512.). Neoepitopes with high affinity (e.g., less than 100nM, less than 75nM, less than 50nM) for previously identified HLA types are then selected for treatment, along with knowledge of the patient's MHC-I/II subtype.

HLA determination can be performed using a variety of methods in wet chemistry known in the art. All of these methods are suitable for use herein. HLA types can be predicted from computational data using reference sequences that contain most or all known and/or common HLA types. For example, the database may provide a relatively large number of patient sequence reads mapped to chromosome 6p21.3 (or any other location near the HLA allele). Most typically, sequence reads will have about 100-300 bases and contain metadata, including read quality, alignment information, orientation, position, and the like. For example, suitable formats include SAM, BAM, FASTA, GAR, and the like. As a non-limiting example, the patient sequence reads may provide a depth of coverage of at least 5x, more typically at least 10x, even more typically at least 20x, and most typically at least 30 x.

In addition to patient sequence reads, the methods of the invention further employ one or more reference sequences comprising sequences of a plurality of known different HLA alleles. For example, a typical reference sequence may be a synthetic (without a corresponding human or other mammalian counterpart) sequence comprising sequence segments of at least one HLA type having multiple HLA alleles of the HLA type. Suitable reference sequences include, but are not limited to, a collection of known genomic sequences of at least 50 different alleles of HLA-a. Alternatively or additionally, the reference sequence may also comprise a collection of known RNA sequences of at least 50 different alleles of HLA-a. The reference sequence is not limited to 50 alleles of HLA-a, but may have alternative compositions with respect to HLA type and number/composition of alleles. Most typically, the reference sequence will be in a computer readable format and will be provided from a database or other data storage device. For example, suitable reference sequence formats include FASTA, FASTQ, EMBL, GCG, or GenBank formats, and may be obtained or constructed directly from data in a common data repository (e.g., IMGT, International ImMunoGeneTics (International ImMunoGeneTics) information system, or Allele Frequency network Database (The Allele Frequency Net Database), eurostat). Alternatively, the reference sequence may also be constructed from individual known HLA alleles based on one or more predetermined criteria (e.g., allele frequency, ethnic allele distribution, common or rare allele type, etc.).

Using the reference sequence, the patient sequence reads can be run through a dibelulin (de Bruijn) map to identify the allele with the best match. Each individual carries two alleles for each HLA type, and these alleles may be very similar, or in some cases even identical. Such high similarity poses a significant problem for conventional alignment schemes. HLA alleles, and even very closely related alleles, can be resolved using a method in which a dibugine map is constructed by breaking down sequence reads into relatively small k-mers (typically of a length between 10-20 bases), and by performing a weighted voting process in which each patient sequence read provides a vote for each allele (a "quantitative read support") based on the k-mer of the sequence read that matches the sequence of the allele. The cumulative highest vote for the allele then indicates the most likely predicted HLA allele. In addition, it is generally preferred that each fragment that matches an allele is also used to calculate the overall coverage and depth of coverage for that allele.

The score can be further refined or refined as needed, especially in cases where many of the top hits (top hits) are similar (e.g., a large portion of their scores are from a highly shared set of k-mers). For example, the score refinement may include a weighting scheme in which alleles that are substantially similar (e.g., > 99%, or other predetermined value) to the current highest hit are removed from future consideration. The count of k-mers used for the current highest hit is then re-weighted by a factor (e.g., 0.5) and the score for each HLA allele is recalculated by adding the weighted counts. This selection process is repeated to find a new highest hit. The accuracy of the method can be even further improved using RNA sequence data that allows identification of alleles expressed by the tumor that may sometimes be only 1 out of 2 alleles present in DNA. DNA or RNA, or a combination of both DNA and RNA, can be processed to make highly accurate HLA predictions, and can be derived from tumor or blood DNA or RNA. In further aspects, suitable methods and considerations for high accuracy in computer modeling of HLA typing are described in WO 2017/035392 (incorporated herein by reference).

Once the patient and tumor specific neoepitopes and HLA types are determined, further computational analysis can be performed by docking the neoepitopes with HLA in silico and determining the best binders (e.g., lowest KD (e.g., less than 500nM or less than 250nM, or less than 150nM, or less than 50nM), e.g., using NetMHC.

After the desired neoepitope is identified, the sequence information of the neoepitope can be used to prepare one or more immunotherapeutic agents. In other agents, a patient may be treated with a virus that is genetically modified with a nucleic acid construct that results in the expression of at least one identified neo-epitope to elicit an immune response against a tumor, as discussed further below. For example, suitable viruses include adenovirus, adeno-associated virus, alphavirus, herpes virus, lentivirus, and the like. However, adenoviruses are particularly preferred. Furthermore, it is further preferred that the virus is a replication-defective non-immunogenic virus, typically achieved by targeted deletion of selected viral proteins (e.g., E1, E3 proteins). Such desirable properties may be further enhanced by deleting the function of the E2b adenovirus gene. High titers of recombinant virus can be achieved using genetically modified human 293 cells (see, e.g., J Virol. [ J. Virol ] (1998)72(2): 926-33).

The virus may be used to infect patient (or non-patient) cells ex vivo or in vivo. For example, the virus may be injected subcutaneously or intravenously, or may be administered intranasally or via inhalation to infect the patient's cells (especially the APCs). Alternatively, immune competent cells (e.g., NK cells, T cells, macrophages, DCs, etc.) can be infected in vitro and then infused into the patient. Alternatively, immunotherapy need not rely on viruses, but may be effected by transfection with nucleic acids or vaccination using RNA or DNA, or other recombinant vectors that result in expression of the neoepitope (e.g., as a single peptide, a tandem minigene, etc.) in the desired cell(s), particularly an immune competent cell.

Most typically, the nucleic acid sequence (for expression from a virally infected cell) is under the control of appropriate regulatory elements well known in the art. For example, suitable promoter elements include constitutively strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK, CAGG promoters), but inducible promoters are also suitable for use herein, particularly under inducing conditions typical for a tumor microenvironment. For example, inducible promoters include those sensitive to hypoxia and those sensitive to TGF- β or IL-8 (e.g., via TRAF, JNK, Erk, or other responsive element promoters). In other examples, suitable inducible promoters include tetracycline inducible promoters, the myxovirus resistance protein 1(Mx1) promoter, and the like.

The manner in which the neoepitopes are arranged and the rationally designed transport of the neoepitopes can affect the efficacy of the immunotherapeutic composition. For example, a single new epitope may be expressed separately from a recombinant construct delivered as a single plasmid, viral expression construct, or the like. Alternatively, multiple neo-epitopes can be expressed separately from separate promoters to form separate mrnas, which are then separately translated into the respective neo-epitopes. A single mRNA containing separate translation initiation points for each new epitope sequence may also be used (e.g., using 2A or IRES signals).

Expression, processing and antigen presentation are considered to be effective where multiple neo-epitopes are expressed from a single transcript to form a single transcript, which is then translated into a single polyepitope. Expression of polyepitopes requires processing by appropriate proteases within the cell (e.g., proteasomes, endosomal proteases, lysosomal proteases) to generate sequences of neoepitopes, and polyepitopes result in improved antigen processing and presentation of most neoepitopes compared to expression of neoepitopes alone, particularly neoepitopes alone in which the length is relatively short (e.g., less than 25 amino acids; results not shown). Moreover, this approach also allows rational design of protease sensitive sequence motifs between the new epitope peptide sequences to ensure or avoid processing by specific proteases (as proteasomes, endosomal proteases and lysosomal proteases have different cleavage preferences). Thus, polyepitopes can be designed that include not only spatially separated linker sequences for new epitopes, but also portions of the sequence (e.g., 3-15 amino acids) that will be preferentially cleaved by specific proteases.

Recombinant nucleic acids and expression vectors (e.g., viral expression vectors) comprising a nucleic acid segment encoding a polyepitope operably coupled to a desired promoter element, and wherein the individual neo-epitopes are optionally separated by a linker and/or a protease cleavage or recognition sequence, may be used. For example, FIG. 11 illustrates various contemplated permutations of neoepitopes for expression from an adenoviral expression system (here: AdV5, with deletions of the E1 and E2b genes). Here, construct 1 illustrates an exemplary neo-epitope arrangement comprising a plurality of neo-epitopes ('minigenes') having a total length of 15 amino acids in the concatemer sequence without an intervening linker sequence, while construct 2 shows the arrangement of construct 1 but comprising nine amino acid linkers between each neo-epitope sequence. Of course, and as noted above, the exact length of the neo-epitope is not limited to 15 amino acids, but may vary considerably. However, in most cases, where additional amino acids are flanked by new epitopes between 8-12 amino acids (e.g., for MHC-I presentation), the total length will typically not exceed 25 amino acids, or 30 amino acids or 50 amino acids. Although fig. 11 indicates a G-S linker, a variety of other linker sequences are also suitable for use herein. Such relatively short neoepitopes are particularly advantageous when the neoepitope is intended to be presented via MHC-I.

A suitable linker sequence will provide spatial flexibility and separate two adjacent new epitopes. However, it is not allowed to select for the linker amino acids which may be immunogenic or which may form an epitope already present in the patient. The polyepitope construct may again be filtered for the presence of epitopes that may be found in the patient (e.g., as part of the normal sequence or due to SNPs or other sequence variations). Such filtering would apply the same techniques and criteria as already discussed above.

Construct 3 illustrates an exemplary neo-epitope arrangement comprising multiple neo-epitopes of a linker sequence that is not inserted in the concatamer sequence, and construct 4 shows the arrangement of construct 3 comprising nine amino acid linkers between each of the neo-epitope sequences. As mentioned above, the exact length of the new epitope is not limited to 25 amino acids, but may vary considerably. However, in most cases, where additional amino acids are flanked by new epitope sequences between 14-20 amino acids (e.g., for MHC-II presentation), the total length will typically not exceed 30 amino acids, or 45 amino acids or 60 amino acids. Although fig. 11 indicates a G-S linker, a variety of other linker sequences are also suitable for use herein. Such relatively short neoepitopes are particularly advantageous when the neoepitope is intended to be presented via MHC-I.

In this example, a 15 amino acid (15-aa) minigene is a MHC class I targeted tumor mutation selected with 7 amino acids of the natural sequence on either side, while a 25 amino acid minigene is a MHC class II targeted tumor mutation selected with 12 amino acids of the natural sequence on either side. An exemplary 9 amino acid linker is of sufficient length to avoid the formation of "non-native" MHC class I epitopes between adjacent minigenes. Multiple epitopes were processed and presented more efficiently than a single neoepitope (data not shown). Adding amino acids of more than 12 amino acids for MHC-I presentation and adding amino acids of more than 20 amino acids for MHC-I presentation improves protease processing.

To maximize intracellular retention of tailored protein sequences for processing and HLA presentation, new epitope sequences can be arranged to minimize hydrophobic sequences, which can refer to trafficking to the cell membrane or extracellular space. Most preferably, detection of the hydrophobic sequence or signal peptide is performed by comparing the sequence to a weight matrix (see, e.g., Nucleic Acids Res. [ Nucleic Acids research ] (1986)14(11):4683-90), or by using a neural network trained on a peptide comprising the signal sequence (see, e.g., J.mol.biol. [ journal of molecular biology ] (2004)338(5): 1027-36). Fig. 12 depicts an exemplary arrangement in which multiple polyepitopes are analyzed. Here, all new bit position permutations are computed to produce a set of permutations. The collection is then processed by a weight matrix and/or neural network prediction to generate a score representing the likelihood of presence and/or strength of a hydrophobic sequence or signal peptide. All positional permutations are then ranked by score, and one or more permutations with scores below a predetermined threshold or lowest score (for the likelihood and/or strength of presence of hydrophobic sequences or signal peptides) are used to construct a customized new epitope expression cassette.

It is generally preferred that the polyepitope comprises at least two, or at least three, or at least five, or at least eight, or at least ten new epitope sequences. Indeed, the payload capacity of recombinant DNA and the availability of filtered and appropriate neo-epitopes are generally considered limiting factors. Thus, adenoviral expression vectors, and particularly Adv5, are particularly preferred because such vectors can accommodate up to 14kb in recombinant payloads.

Neoepitopes/polyepitopes can be targeted to specific subcellular compartments (e.g., cytosol, endosomes, lysosomes) and thus to specific MHC presentation types. Such targeted expression, processing and presentation is particularly advantageous because it can be prepared to direct the immune response to CD8⁺Type response (in which multiple epitopes are targeted to the cytoplasmic space) or to CD4⁺Compositions of type response (wherein multiple epitopes are targeted to endosomal/lysosomal compartments). Polyepitopes that are normally presented via the MHC-I pathway can be presented via the MHC-II pathway (and thus mimic cross presentation of neoepitopes). Neoepitopes and polyepitopic sequences can be designed and directed to one or two MHC presentation pathways using appropriate sequence elements. MHC-I presented peptides will typically be produced from the cytoplasm via proteasome processing and delivered through the endoplasmic reticulum. Thus, as discussed in more detail below, expression of epitopes intended for MHC-I presentation will generally be directed to the cytoplasm. On the other hand, MHC-II presented peptides will typically be produced from endosomal and lysosomal compartments via degradation and processing of acid proteases (e.g., legumain, cathepsin L, and cathepsin S) prior to delivery to the cell membrane.

Polyepitopic protein degradation may also be enhanced using a variety of methods, including the addition of a cleavable or non-cleavable ubiquitin moiety to the N-terminus, and/or the placement of one or more destabilizing amino acids (e.g., N, K, C, F, E, R, Q) at the N-terminus of the polyepitopic, where presentation is directed to MHC-I. In cases where presentation is directed to MHC-II, the cleavage site for endosomal or lysosomal proteases can be engineered as a polyepitope.

The signal and/or leader peptide can transport the neoepitope and/or polyepitope to endosomal and lysosomal compartments, or retain the neoepitope/polyepitope in the cytoplasmic space. For example, to export the polyepitope to an endosome or lysosome, a leader peptide (e.g., CD1b leader peptide) can sequester the polyepitope from the cytoplasm. Additionally or alternatively, targeting pro-sequences and/or targeting peptides may be added to the N-terminus and/or C-terminus. Targeting pro-sequences typically comprise between 6 and 136 basic amino acids and hydrophobic amino acids. The sequence for peroxisome targeting may be at the C-terminus. Other signals (e.g., signal spots) include sequence elements that are separated in the peptide sequence and function after appropriate peptide folding. Protein modifications (like glycosylation) can induce targeting. Suitable targeting signals include, but are not limited to, peroxisome targeting signal 1(PTS1) and peroxisome targeting signal 2(PTS 2).

Furthermore, proteins can be sorted into endosomes and lysosomes by signals within the cytosolic domain of the protein, which are typically short linear sequences. "tyrosine-based" sorting signals correspond to NPXY or

Consensus motifs. "Dual leucine-based" signals are suitable [ DE]XXXL[LI]Or dxll consensus motif. All these signals are recognized by protein envelope components on the cytosolic surface of the membrane. Adapter Protein (AP) complexes AP-1, AP-2, AP-3 and AP-4 recognize proteins with characteristic fine specificity

And [ DE]XXXL[LI]Signals, however, the GGA adaptor family recognizes dxll signals. The "FYVE" domain is associated with vacuolar protein sorting and endosomal function. Human CD1 tail sequence (see, e.g., Immunology [. Immunology ]]122:522-31) can also target endosomes. The LAMP1-TM (transmembrane) domain targets lysosomes. The CD1a tail sequence targets the recycling endosomes. The Cd1c tail sequence was targeted to sorting endosomes.

The polyepitope may be a chimeric polyepitope that includes at least a portion, and more typically the entire TAA (e.g., CEA, PSMA, PSA, MUC1, AFP, MAGE, HER2, HCC1, p62, p90, etc.). TAA is usually processed and presented via MHC-II. Thus, instead of using compartment-specific signals and/or leader sequences, the processing machinery for TAAs can use MHC-II targeting.

Transport to or retention in the cytosolic compartment does not necessarily require one or more specific sequence elements. However, N-terminal or C-terminal cytoplasmic retention signals (e.g., SNAP-25, syntaxin, synapsin, synaptomains, vesicle-associated membrane protein (VAMP), synaptobrevin (SV2), high affinity choline transporters, neurotoxins, voltage-gated calcium channels, acetylcholinesterase, and NOTCH) can be added, including membrane-anchoring proteins or membrane-anchoring domains of membrane-anchoring proteins.

The polyepitope may also comprise one or more transmembrane segments to direct the neoepitope outside the cell after processing, thereby making it visible to immune competent cells. Many transmembrane domains are known in the art, all of which are suitable for use herein, including those having a single alpha helix, multiple alpha helices, an alpha/beta barrel configuration, and the like. For example, contemplated transmembrane domains include, but are not limited to, T cell receptor, CD epsilon, CD (e.g., CD α, CD β), CD, OX, CD134, CD137, CD154, KIRDS, OX, CD, LFA-1(CD11, CD), ICOS (CD278), 4-1BB (CD137), GITR, CD, BAFFR, HVEM (LIGHTR), SLAMF, NKp (KLRF), CD160, CD, IL2 β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, ITGAA-1, ITGAM, CD11, ITGAX, CD11, ITGB, LFGB, CD160, ITGAR-160, ACAR, CD103, ITGAM (SLAMF), SLAM-100, CD-100, ITGAM, CD-6, ITGAD, ITGAE, CD-6, ITGAE, CD-, BLAME (SLAMF8), SELPLG (CD162), LTBR or PAG/one or more transmembrane regions of the alpha, beta or zeta chain of Cbp. Where a fusion protein is desired, the recombinant chimeric gene may have a first portion encoding one or more transmembrane regions and a second portion encoding a suppressor protein (which is in-frame with the first portion). This would not achieve presentation of MHC complexes and thus provide for neo-epitope presentation independent of MHC/T cell receptor interactions, which may open an additional pathway for immune recognition to trigger antibody production against the neo-epitope.

Alternatively or additionally, the polyepitope may also include an export signal sequence, forcing the transfected cells to produce and secrete one or more neo-epitopes. For example, the addition of the SPARC leader sequence to a neoepitope or polyepitope sequence can enable secretion of the neoepitope/polyepitope into the extracellular space in vivo. Advantageously, such secreted neoepitope or polyepitope is then taken up by immune competent cells (especially APCs, e.g. DCs), which typically process and display the neoepitope via the MHC-II pathway.

Alternatively or additionally, the neo-epitope or polyepitope may be administered as a peptide, optionally in association with a carrier protein. Among other suitable carrier proteins, human albumin or lactoferrin are preferred. The carrier Protein may be in a native conformation, or pre-treated to form nanoparticles with exposed hydrophobic domains (see, e.g., (2015) Adv Protein Chem Struct Biol. [ advances in Protein chemistry and structure biology ]98:121-43), and a neo-epitope or multi-epitopes may be conjugated to these carrier proteins. Most typically, the neoepitope or polyepitopes are non-covalently coupled to a carrier protein. The carrier-bound neo-epitope or polyepitopes will be taken up by immune competent cells (especially APCs, e.g., DCs), which typically process and display the neo-epitope via the MHC-II pathway.

Immunotherapeutic compositions can deliver one or more neo-epitopes to various subcellular locations, resulting in different immune responses. For example, prior art figure 13 illustrates that polyepitopes are processed primarily in the proteasome and are presented via MHC-I. MHC antigen is expressed by CD8⁺T cell recognition. Thus, targeting multiple epitope processing to the cytosol would direct the immune response to CD8⁺The response is skewed. Prior art FIG. 14, on the other hand, illustrates that polyepitopes are processed primarily in the endosome and presented via MHC-II. In this case, the MHC antigen is expressed by CD4⁺T cell recognition. Thus, targeting polyepitope processing to endosomes or lysosomes would direct the immune response to CD4⁺The response is skewed. Such targeting methods deliver polyepitope and neo-epitope peptides to a particular MHC subtype with the highest affinity for the peptide, even though the peptides will not be presented by the MHC subtype. In the examples below, additional added amino acids allow processing flexibility in the cytoplasm, proteasome, and endosomes.

Neo-epitopes or multi-epitope trafficking patterns can be combined to suit a particular purpose. For example, sequential administration of the same neoepitope or polyepitopes with different targets may function in a prime-boost regimen. Upon first administration, the patient is vaccinated with the recombinant virus to infect the patient's cells, resulting in antigen expression, processing and MHC-I presentation, thereby effecting a first immune response derived intracellularly. Then, a second administration of the same neoepitope bound to albumin can boost immunity because the APC presents the protein on MHC-II. The same neoepitope or polyepitopes transported for MHC independent presentation for cell surface binding may promote ADCC response or NK-mediated cell killing. As illustrated in the examples below, cross-presentation or MHC-II directed presentation can enhance the immunogenicity of the neoepitope.

Multiple different trafficking of the same neoepitope or polyepitopes can be achieved in many ways. For example, different trafficked neo-epitopes or polyepitopes can be administered separately, using the same (e.g., viral expression vector) or different (e.g., viral expression vector and albumin binding) patterns. Similarly, and especially when the therapeutic agent is an expression system (e.g., viral or bacterial), the recombinant nucleic acid can comprise two different portions that encode the same, though differently trafficked neo-or polyepitope (e.g., a first portion trafficked to a first location (e.g., cytosolic or endosomal or lysosomal), a second portion trafficked to a second, different location (e.g., cytosolic or endosomal or lysosomal, secreted, membrane bound)). Likewise, a first administration may target the neoepitope or polyepitope to the cytoplasm, while a second administration (typically at least one, two, four, one or two days after the first administration) may target the neoepitope or polyepitope to endosomes or lysosomes, or secrete them outside the cell.

An exemplary arrangement of neoepitopes and protein adjuvants is depicted in fig. 15. Here, the recombinant nucleic acid encodes a first series of neo-epitopes coupled together by linkers. This first segment is coupled to a second series of neo-epitopes coupled together by respective linkers. Between the first and second segments is the self-cleaving peptide sequence of GSG-P2A. Downstream of the second series of neoepitopes are segments encoding two separate costimulatory molecules, followed by segments encoding checkpoint inhibitors. Further downstream of the checkpoint inhibitor coding sequence is a segment encoding an adjuvant peptide. The arrangement of fig. 15 is merely illustrative. Other arrangements and content are also suitable.

Although not shown in FIG. 15, the inclusion of the human CD74 derivative sequence element "Ii-bond (Ii-key)" in the recombinant nucleic acid may increase the immunogenicity of MHC-II presented epitopes. Exemplary sequence elements include "LRMKLPKPPKPVSKMR" and shorter versions thereof, particularly "LRMK". Such sequence elements may be placed 5' to one or more patient and/or tumor specific neo-epitopes. For example, the construct may contain one or more "Ii-bond" sequences in the MHC II targeting polyepitope, optionally with one or more intra-epitope linkers (GPGPG-LRMK) to enhance each epitope, immediately following the leader peptide sequence and preceding the polyepitope sequence.

The expression construct (e.g., expression vector or plasmid) may further encode at least one, at least two, at least three, or even at least four co-stimulatory molecules to enhance the interaction between the infected cell (e.g., APC) and the T cell. Non-limiting examples include CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, or even GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, LFA3, and SLAM family members. Particularly preferred molecules for coordinated expression include CD80(B7-1), CD86(B7-2), CD54(ICAM-1) and CD11 (LFA-1). One or more cytokines or cytokine analogs may also be expressed from the recombinant nucleic acid. Non-limiting examples include IL-2, IL-15, and IL-15 superagonists (ALT-803). Expression of the co-stimulatory molecules and/or cytokines may be coordinated such that the neoepitope or polyepitope is expressed simultaneously with the co-stimulatory molecules and/or cytokines. Co-stimulatory molecules and/or cytokines may be produced from a single transcript (optionally including a multi-epitope coding sequence), for example using IRES or 2A sequences, or from multiple transcripts.

The viral vector may also encode one or more checkpoint receptor ligands. Most typically, binding inhibits checkpoint signaling. Non-limiting examples of receptors include CTLA-4 (particularly against CD 8)⁺Cells), PD-1 (especially against CD 4)⁺Cells), TIM1 receptor, 2B4, and CD 160. Suitable peptide conjugates may include antibody fragments, particularly scfvs. Small molecule peptide ligands that specifically bind to the receptor (e.g., isolated via RNA display or phage panning) are also useful. The expression of checkpoint inhibitors can be coordinated such that neo-epitopes or polyepitopes are expressed simultaneously. Ligands can be generated from a single transcript (optionally including multi-epitope coding sequences), for example using IRES or 2A sequences, or from multiple transcripts.

All of the above co-stimulators and checkpoint inhibitors are well known in the art, and sequence information for genes, isoforms, and variants encoding these proteins can be retrieved from a variety of public sources, including sequence databases accessible at NCBI, EMBL, GenBank, RefSeq, and the like. Although the above exemplary stimulatory molecules may be expressed in full-length human forms, modified and non-human forms are also suitable, so long as such forms stimulate or activate T cells. Thus, muteins, truncates and chimeras are also suitable.

The expression construct preferably comprises a sequence encoding one or more polyepitopes, wherein at least one, at least two, or all of the polyepitopes comprise trafficking signals directing the polyepitope to at least one, and more typically at least two, subcellular locations. For example, a first polyepitope can be transported to the cytoplasm, while a second polyepitope can be transported to an endosome or lysosome. Alternatively, the first polyepitope may be transported to an endosome or lysosome, while the second polyepitope is transported to the cell membrane or secretions.

Viral expression constructs (e.g., adenoviruses, particularly Δ E1/Δ E2b AdV5) can be used alone or in combination as therapeutic vaccines for treatment with allogeneic or autologous natural killer or T cell therapy-either in naked form or carrying chimeric antigen receptors expressing antibodies targeting neoepitopes, tumor-associated antigens, or the same payload as the virus. Natural killer cells, including patient-derived NK-92 cell lines, may also express CD16 and may be conjugated to antibodies.

Additional therapeutic neoepitope-based modalities (e.g., synthetic antibodies against neoepitopes as described in WO 2016/172722) can be administered alone or in combination with autologous or allogeneic NK cells, particularly haNK cells or taNK cells (e.g., both commercially available from north-yerster, NantKwest, 9920 jackson great street, karl City, 90232, ca). As non-limiting examples, haNK cells may carry recombinant antibodies on CD16 variants that bind to the neo-epitope of the treated patient, and taNK cells may carry chimeric antigen receptors that bind to the neo-epitope of the treated patient. Additional treatment modalities may also be independent of neoepitopes, such as activated NK cells (e.g., aNK cells, commercially available from north tery, 9920 jackson great street, karuflex city, ca 90232), as well as non-cell based therapies (e.g., chemotherapy and/or radiation therapy). Immunostimulatory cytokines, particularly IL-2, IL15, and IL-21, may be administered alone or in combination with one or more checkpoint inhibitors (e.g., ipilimumab, nivolumab, etc.). Additional pharmaceutical intervention may include administration of one or more drugs that suppress immunosuppressive cells, especially MDSCs, tregs, and M2 macrophages. Suitable drugs for this purpose include inhibitors of IL-8 or interferon-gamma or antibodies that bind IL-8 or interferon-gamma; and agents that inactivate MDSCs (e.g., NO inhibitors, arginase inhibitors, ROS inhibitors); drugs that block development or differentiate into MDSCs (e.g., IL-12, VEGF inhibitors, bisphosphonates); or an agent that is toxic to MDSCs (e.g., gemcitabine, cisplatin, 5-FU). Similarly, cyclophosphamide, daclizumab, and anti-GITR or anti-OX 40 antibodies can inhibit tregs.

Chemotherapy and/or radiation therapy at low doses, preferably in a rhythmic regime, can trigger the overexpression or transcription of stress signals. Such treatments may use doses that affect protein expression, cell division and/or cell cycle, preferably induce apoptosis or stress-related genes (in particular NKG2D ligands). Such treatment may include low dose treatment with one or more chemotherapeutic agents. Most typically, the low dose therapeutic exposure should be the LD of the chemotherapeutic agent₅₀Or IC₅₀Not more than 70%, equal to or less than 50%, equal to or less than 40%, equal to or less than 30%, equal to or less than 20%, equal to or less than 10%, or equal to or less than 5%. Such low dose regimens may be performed in a rhythmic manner as described in US 7,758,891, US 7,771,751, US 7,780,984, US 7,981,445 and US 8,034,375.

All known chemotherapeutic agents are suitable for use in the methods disclosed herein, including by way of non-limiting example kinase inhibitors, receptor agonists and antagonists, antimetabolites, cytostatic and cytotoxic agents. Pathway analysis of the physiological data can be used to identify drugs suitable for interfering with or inhibiting pathways driving tumor growth or development, as described in WO 11/139345 and WO 13/62505. Expression of stress-related genes in tumor cells drives surface presentation of NKG2D, NKP30, NKP44 and/or NKP46 ligands, which activate NK cells to destroy tumor cells. Low dose chemotherapy can trigger tumor cells to express and display stress-associated proteins.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. Numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

Unless the context indicates to the contrary, all ranges set forth herein are to be construed as including their endpoints, and open-ended ranges are to be construed as including only commercially practical values. Similarly, a list of all values should be considered to include intermediate values unless the context indicates the contrary. As used herein in the specification and throughout the claims that follow, the meaning of "a", "an" and "the" includes plural references unless the context clearly dictates otherwise. Also, as used in the specification herein, the meaning of "in … …" includes "in … …" and "on … …" unless the context clearly dictates otherwise.

It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the concepts disclosed herein. The claimed subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises/comprising" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the claims recite at least one of something selected from the group consisting of A, B, C … … and N, the word should be construed to require only one element of the group, rather than A plus N, or B plus N, etc.

Sequence listing

<110> Nante cell Co., Ltd (Nantcell)

<120> CD40 and CD40L conjugates in adenoviral vaccine vehicle

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Asn Leu His Glu Asp Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn

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Gln Phe Glu Gly Phe Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr

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Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val

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Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val

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Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr

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Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser

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Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe

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Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro

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Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro

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Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His

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Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser

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Gly Gly Gly Gly Ser Gly Gly Gly Pro Pro Thr Ala Cys Arg Glu Lys

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Gln Tyr Leu Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln

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Lys Leu Val Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro

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Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys

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His Gln His Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln

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Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp

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His Cys Thr Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys

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Ser Pro Gly Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr

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Ile Cys Glu Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala

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Phe Glu Lys Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val

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Val Gln Gln Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln

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Asp Arg Leu Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu

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Phe Ala Ile Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys

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Pro Thr Asn Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn

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Phe Pro Asp Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu

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Thr Leu His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser

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Arg Ile Ser Val Gln Glu Arg Gln

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Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

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Ala Val His Pro Glu His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg

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Asn Leu His Glu Asp Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn

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Thr Gly Glu Arg Ser Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser

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Gln Phe Glu Gly Phe Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr

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Lys Lys Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln

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Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val

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Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val

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Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr

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Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser

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Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe

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Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro

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Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro

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Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His

210 215 220

Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Pro Thr Ala Cys Arg

245 250 255

Glu Lys Gln Tyr Leu Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro

260 265 270

Gly Gln Lys Leu Val Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys

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Leu Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr

290 295 300

His Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val

305 310 315 320

Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu

325 330 335

Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg

340 345 350

Ser Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser

355 360 365

Asp Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser

370 375 380

Ser Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp

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Leu Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly

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Pro Gln Asp Arg Leu Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly

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Ile Leu Phe Ala Ile Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala

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Lys Lys Pro Thr Asn Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu

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Ile Asn Phe Pro Asp Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val

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Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys

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Glu Ser Arg Ile Ser Val Gln Glu Arg Gln

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Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Pro Glu His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg

20 25 30

Asn Leu His Glu Asp Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn

35 40 45

Thr Gly Glu Arg Ser Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser

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Gln Phe Glu Gly Phe Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr

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Lys Lys Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln

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Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val

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Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val

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Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr

130 135 140

Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser

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Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe

165 170 175

Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro

180 185 190

Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro

195 200 205

Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His

210 215 220

Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro Pro Thr Ala

245 250 255

Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln Cys Cys Ser Leu Cys

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Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr Glu Phe Thr Glu Thr

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Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp Asn Arg

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Glu Thr His Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu Gly Leu

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Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys Thr Cys

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Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys Val Leu

340 345 350

His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala Thr Gly

355 360 365

Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe Ser Asn

370 375 380

Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys Glu Thr

385 390 395 400

Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp Val Val

405 410 415

Cys Gly Pro Gln Asp Arg Leu Arg Ala Leu Val Val Ile Pro Ile Ile

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Phe Gly Ile Leu Phe Ala Ile Leu Leu Val Leu Val Phe Ile Lys Lys

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Val Ala Lys Lys Pro Thr Asn Lys Ala Pro His Pro Lys Gln Glu Pro

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Gln Glu Ile Asn Phe Pro Asp Asp Leu Pro Gly Ser Asn Thr Ala Ala

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Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln Glu Asp

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Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln

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Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

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Ala Val His Pro Glu His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg

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Asn Leu His Glu Asp Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn

35 40 45

Thr Gly Glu Arg Ser Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser

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Gln Phe Glu Gly Phe Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr

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Lys Lys Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln

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Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val

100 105 110

Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val

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Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr

130 135 140

Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser

145 150 155 160

Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe

165 170 175

Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro

180 185 190

Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro

195 200 205

Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His

210 215 220

Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Pro Pro

245 250 255

Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln Cys Cys Ser

260 265 270

Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr Glu Phe Thr

275 280 285

Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp

290 295 300

Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu

305 310 315 320

Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys

325 330 335

Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys

340 345 350

Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala

355 360 365

Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe

370 375 380

Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys

385 390 395 400

Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp

405 410 415

Val Val Cys Gly Pro Gln Asp Arg Leu Arg Ala Leu Val Val Ile Pro

420 425 430

Ile Ile Phe Gly Ile Leu Phe Ala Ile Leu Leu Val Leu Val Phe Ile

435 440 445

Lys Lys Val Ala Lys Lys Pro Thr Asn Lys Ala Pro His Pro Lys Gln

450 455 460

Glu Pro Gln Glu Ile Asn Phe Pro Asp Asp Leu Pro Gly Ser Asn Thr

465 470 475 480

Ala Ala Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln

485 490 495

Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln

500 505 510

<210> 5

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<212> PRT

<213> Artificial sequence

<220>

<223> CD40/CD40L +20mer linker

<400> 5

Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Pro Glu His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg

20 25 30

Asn Leu His Glu Asp Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn

35 40 45

Thr Gly Glu Arg Ser Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser

50 55 60

Gln Phe Glu Gly Phe Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr

65 70 75 80

Lys Lys Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln

85 90 95

Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val

100 105 110

Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val

115 120 125

Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr

130 135 140

Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser

145 150 155 160

Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe

165 170 175

Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro

180 185 190

Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro

195 200 205

Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His

210 215 220

Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln Cys

260 265 270

Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr Glu

275 280 285

Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu Asp

290 295 300

Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp Pro

305 310 315 320

Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr

325 330 335

Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys Glu

340 345 350

Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys Gln

355 360 365

Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val Gly

370 375 380

Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp Thr

385 390 395 400

Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn Lys

405 410 415

Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg Ala Leu Val Val

420 425 430

Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile Leu Leu Val Leu Val

435 440 445

Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn Lys Ala Pro His Pro

450 455 460

Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp Asp Leu Pro Gly Ser

465 470 475 480

Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro Val

485 490 495

Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln

500 505 510

<210> 6

<211> 515

<212> PRT

<213> Artificial sequence

<220>

<223> mouse _ CD40/CD40L +12mer linker

<400> 6

Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Leu His Arg Arg Leu Asp Lys Val Glu Glu Glu Val Asn

20 25 30

Leu His Glu Asp Phe Val Phe Ile Lys Lys Leu Lys Arg Cys Asn Lys

35 40 45

Gly Glu Gly Ser Leu Ser Leu Leu Asn Cys Glu Glu Met Arg Arg Gln

50 55 60

Phe Glu Asp Leu Val Lys Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys

65 70 75 80

Glu Asn Ser Phe Glu Met Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala

85 90 95

Ala His Val Val Ser Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln

100 105 110

Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu

115 120 125

Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val

130 135 140

Tyr Thr Gln Val Thr Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg

145 150 155 160

Pro Phe Ile Val Gly Leu Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg

165 170 175

Ile Leu Leu Lys Ala Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu

180 185 190

Gln Gln Ser Val His Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala

195 200 205

Ser Val Phe Val Asn Val Thr Glu Ala Ser Gln Val Ile His Arg Val

210 215 220

Gly Phe Ser Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gly Gly Gly Gly Gln Cys Val Thr Cys Ser Asp Lys Gln

245 250 255

Tyr Leu His Asp Gly Gln Cys Cys Asp Leu Cys Gln Pro Gly Ser Arg

260 265 270

Leu Thr Ser His Cys Thr Ala Leu Glu Lys Thr Gln Cys His Pro Cys

275 280 285

Asp Ser Gly Glu Phe Ser Ala Gln Trp Asn Arg Glu Ile Arg Cys His

290 295 300

Gln His Arg His Cys Glu Pro Asn Gln Gly Leu Arg Val Lys Lys Glu

305 310 315 320

Gly Thr Ala Glu Ser Asp Thr Val Cys Thr Cys Lys Glu Gly Gln His

325 330 335

Cys Thr Ser Lys Asp Cys Glu Ala Cys Ala Gln His Thr Pro Cys Ile

340 345 350

Pro Gly Phe Gly Val Met Glu Met Ala Thr Glu Thr Thr Asp Thr Val

355 360 365

Cys His Pro Cys Pro Val Gly Phe Phe Ser Asn Gln Ser Ser Leu Phe

370 375 380

Glu Lys Cys Tyr Pro Trp Thr Ser Cys Glu Asp Lys Asn Leu Glu Val

385 390 395 400

Leu Gln Lys Gly Thr Ser Gln Thr Asn Val Ile Cys Gly Leu Lys Ser

405 410 415

Arg Met Arg Ala Leu Leu Val Ile Pro Val Val Met Gly Ile Leu Ile

420 425 430

Thr Ile Phe Gly Val Phe Leu Tyr Ile Lys Lys Val Val Lys Lys Pro

435 440 445

Lys Asp Asn Glu Ile Leu Pro Pro Ala Ala Arg Arg Gln Asp Pro Gln

450 455 460

Glu Met Glu Asp Tyr Pro Gly His Asn Thr Ala Ala Pro Val Gln Glu

465 470 475 480

Thr Leu His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser

485 490 495

Arg Ile Ser Val Gln Glu Arg Gln Val Thr Asp Ser Ile Ala Leu Arg

500 505 510

Pro Leu Val

515

<210> 7

<211> 517

<212> PRT

<213> Artificial sequence

<220>

<223> mouse _ CD40/CD40L +14mer linker

<400> 7

Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Leu His Arg Arg Leu Asp Lys Val Glu Glu Glu Val Asn

20 25 30

Leu His Glu Asp Phe Val Phe Ile Lys Lys Leu Lys Arg Cys Asn Lys

35 40 45

Gly Glu Gly Ser Leu Ser Leu Leu Asn Cys Glu Glu Met Arg Arg Gln

50 55 60

Phe Glu Asp Leu Val Lys Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys

65 70 75 80

Glu Asn Ser Phe Glu Met Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala

85 90 95

Ala His Val Val Ser Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln

100 105 110

Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu

115 120 125

Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val

130 135 140

Tyr Thr Gln Val Thr Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg

145 150 155 160

Pro Phe Ile Val Gly Leu Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg

165 170 175

Ile Leu Leu Lys Ala Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu

180 185 190

Gln Gln Ser Val His Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala

195 200 205

Ser Val Phe Val Asn Val Thr Glu Ala Ser Gln Val Ile His Arg Val

210 215 220

Gly Phe Ser Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Gly Gln Cys Val Thr Cys Ser Asp

245 250 255

Lys Gln Tyr Leu His Asp Gly Gln Cys Cys Asp Leu Cys Gln Pro Gly

260 265 270

Ser Arg Leu Thr Ser His Cys Thr Ala Leu Glu Lys Thr Gln Cys His

275 280 285

Pro Cys Asp Ser Gly Glu Phe Ser Ala Gln Trp Asn Arg Glu Ile Arg

290 295 300

Cys His Gln His Arg His Cys Glu Pro Asn Gln Gly Leu Arg Val Lys

305 310 315 320

Lys Glu Gly Thr Ala Glu Ser Asp Thr Val Cys Thr Cys Lys Glu Gly

325 330 335

Gln His Cys Thr Ser Lys Asp Cys Glu Ala Cys Ala Gln His Thr Pro

340 345 350

Cys Ile Pro Gly Phe Gly Val Met Glu Met Ala Thr Glu Thr Thr Asp

355 360 365

Thr Val Cys His Pro Cys Pro Val Gly Phe Phe Ser Asn Gln Ser Ser

370 375 380

Leu Phe Glu Lys Cys Tyr Pro Trp Thr Ser Cys Glu Asp Lys Asn Leu

385 390 395 400

Glu Val Leu Gln Lys Gly Thr Ser Gln Thr Asn Val Ile Cys Gly Leu

405 410 415

Lys Ser Arg Met Arg Ala Leu Leu Val Ile Pro Val Val Met Gly Ile

420 425 430

Leu Ile Thr Ile Phe Gly Val Phe Leu Tyr Ile Lys Lys Val Val Lys

435 440 445

Lys Pro Lys Asp Asn Glu Ile Leu Pro Pro Ala Ala Arg Arg Gln Asp

450 455 460

Pro Gln Glu Met Glu Asp Tyr Pro Gly His Asn Thr Ala Ala Pro Val

465 470 475 480

Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys

485 490 495

Glu Ser Arg Ile Ser Val Gln Glu Arg Gln Val Thr Asp Ser Ile Ala

500 505 510

Leu Arg Pro Leu Val

515

<210> 8

<211> 519

<212> PRT

<213> Artificial sequence

<220>

<223> mouse _ CD40/CD40L +16mer linker

<400> 8

Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Leu His Arg Arg Leu Asp Lys Val Glu Glu Glu Val Asn

20 25 30

Leu His Glu Asp Phe Val Phe Ile Lys Lys Leu Lys Arg Cys Asn Lys

35 40 45

Gly Glu Gly Ser Leu Ser Leu Leu Asn Cys Glu Glu Met Arg Arg Gln

50 55 60

Phe Glu Asp Leu Val Lys Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys

65 70 75 80

Glu Asn Ser Phe Glu Met Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala

85 90 95

Ala His Val Val Ser Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln

100 105 110

Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu

115 120 125

Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val

130 135 140

Tyr Thr Gln Val Thr Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg

145 150 155 160

Pro Phe Ile Val Gly Leu Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg

165 170 175

Ile Leu Leu Lys Ala Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu

180 185 190

Gln Gln Ser Val His Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala

195 200 205

Ser Val Phe Val Asn Val Thr Glu Ala Ser Gln Val Ile His Arg Val

210 215 220

Gly Phe Ser Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gln Cys Val Thr Cys

245 250 255

Ser Asp Lys Gln Tyr Leu His Asp Gly Gln Cys Cys Asp Leu Cys Gln

260 265 270

Pro Gly Ser Arg Leu Thr Ser His Cys Thr Ala Leu Glu Lys Thr Gln

275 280 285

Cys His Pro Cys Asp Ser Gly Glu Phe Ser Ala Gln Trp Asn Arg Glu

290 295 300

Ile Arg Cys His Gln His Arg His Cys Glu Pro Asn Gln Gly Leu Arg

305 310 315 320

Val Lys Lys Glu Gly Thr Ala Glu Ser Asp Thr Val Cys Thr Cys Lys

325 330 335

Glu Gly Gln His Cys Thr Ser Lys Asp Cys Glu Ala Cys Ala Gln His

340 345 350

Thr Pro Cys Ile Pro Gly Phe Gly Val Met Glu Met Ala Thr Glu Thr

355 360 365

Thr Asp Thr Val Cys His Pro Cys Pro Val Gly Phe Phe Ser Asn Gln

370 375 380

Ser Ser Leu Phe Glu Lys Cys Tyr Pro Trp Thr Ser Cys Glu Asp Lys

385 390 395 400

Asn Leu Glu Val Leu Gln Lys Gly Thr Ser Gln Thr Asn Val Ile Cys

405 410 415

Gly Leu Lys Ser Arg Met Arg Ala Leu Leu Val Ile Pro Val Val Met

420 425 430

Gly Ile Leu Ile Thr Ile Phe Gly Val Phe Leu Tyr Ile Lys Lys Val

435 440 445

Val Lys Lys Pro Lys Asp Asn Glu Ile Leu Pro Pro Ala Ala Arg Arg

450 455 460

Gln Asp Pro Gln Glu Met Glu Asp Tyr Pro Gly His Asn Thr Ala Ala

465 470 475 480

Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln Glu Asp

485 490 495

Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln Val Thr Asp Ser

500 505 510

Ile Ala Leu Arg Pro Leu Val

515

<210> 9

<211> 521

<212> PRT

<213> Artificial sequence

<220>

<223> mouse _ CD40/CD40L +18mer linker

<400> 9

Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Leu His Arg Arg Leu Asp Lys Val Glu Glu Glu Val Asn

20 25 30

Leu His Glu Asp Phe Val Phe Ile Lys Lys Leu Lys Arg Cys Asn Lys

35 40 45

Gly Glu Gly Ser Leu Ser Leu Leu Asn Cys Glu Glu Met Arg Arg Gln

50 55 60

Phe Glu Asp Leu Val Lys Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys

65 70 75 80

Glu Asn Ser Phe Glu Met Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala

85 90 95

Ala His Val Val Ser Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln

100 105 110

Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu

115 120 125

Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val

130 135 140

Tyr Thr Gln Val Thr Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg

145 150 155 160

Pro Phe Ile Val Gly Leu Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg

165 170 175

Ile Leu Leu Lys Ala Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu

180 185 190

Gln Gln Ser Val His Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala

195 200 205

Ser Val Phe Val Asn Val Thr Glu Ala Ser Gln Val Ile His Arg Val

210 215 220

Gly Phe Ser Ser Phe Gly Leu Leu Lys Leu Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gln Cys Val

245 250 255

Thr Cys Ser Asp Lys Gln Tyr Leu His Asp Gly Gln Cys Cys Asp Leu

260 265 270

Cys Gln Pro Gly Ser Arg Leu Thr Ser His Cys Thr Ala Leu Glu Lys

275 280 285

Thr Gln Cys His Pro Cys Asp Ser Gly Glu Phe Ser Ala Gln Trp Asn

290 295 300

Arg Glu Ile Arg Cys His Gln His Arg His Cys Glu Pro Asn Gln Gly

305 310 315 320

Leu Arg Val Lys Lys Glu Gly Thr Ala Glu Ser Asp Thr Val Cys Thr

325 330 335

Cys Lys Glu Gly Gln His Cys Thr Ser Lys Asp Cys Glu Ala Cys Ala

340 345 350

Gln His Thr Pro Cys Ile Pro Gly Phe Gly Val Met Glu Met Ala Thr

355 360 365

Glu Thr Thr Asp Thr Val Cys His Pro Cys Pro Val Gly Phe Phe Ser

370 375 380

Asn Gln Ser Ser Leu Phe Glu Lys Cys Tyr Pro Trp Thr Ser Cys Glu

385 390 395 400

Asp Lys Asn Leu Glu Val Leu Gln Lys Gly Thr Ser Gln Thr Asn Val

405 410 415

Ile Cys Gly Leu Lys Ser Arg Met Arg Ala Leu Leu Val Ile Pro Val

420 425 430

Val Met Gly Ile Leu Ile Thr Ile Phe Gly Val Phe Leu Tyr Ile Lys

435 440 445

Lys Val Val Lys Lys Pro Lys Asp Asn Glu Ile Leu Pro Pro Ala Ala

450 455 460

Arg Arg Gln Asp Pro Gln Glu Met Glu Asp Tyr Pro Gly His Asn Thr

465 470 475 480

Ala Ala Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln

485 490 495

Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln Val Thr

500 505 510

Asp Ser Ile Ala Leu Arg Pro Leu Val

515 520

<210> 10

<211> 523

<212> PRT

<213> Artificial sequence

<220>

<223> mouse _ CD40/CD40L +20mer linker

<400> 10

Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Leu His Arg Arg Leu Asp Lys Val Glu Glu Glu Val Asn

20 25 30

Leu His Glu Asp Phe Val Phe Ile Lys Lys Leu Lys Arg Cys Asn Lys

35 40 45

Gly Glu Gly Ser Leu Ser Leu Leu Asn Cys Glu Glu Met Arg Arg Gln

50 55 60

Phe Glu Asp Leu Val Lys Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys

65 70 75 80

Glu Asn Ser Phe Glu Met Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala

85 90 95

Ala His Val Val Ser Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln

100 105 110

Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu

115 120 125

Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val

130 135 140

Tyr Thr Gln Val Thr Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg

145 150 155 160

Pro Phe Ile Val Gly Leu Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg

165 170 175

Ile Leu Leu Lys Ala Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu

180 185 190

Gln Gln Ser Val His Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala

195 200 205

Ser Val Phe Val Asn Val Thr Glu Ala Ser Gln Val Ile His Arg Val

210 215 220

Gly Phe Ser Ser Phe Gly Leu Leu Lys Leu Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gln

245 250 255

Cys Val Thr Cys Ser Asp Lys Gln Tyr Leu His Asp Gly Gln Cys Cys

260 265 270

Asp Leu Cys Gln Pro Gly Ser Arg Leu Thr Ser His Cys Thr Ala Leu

275 280 285

Glu Lys Thr Gln Cys His Pro Cys Asp Ser Gly Glu Phe Ser Ala Gln

290 295 300

Trp Asn Arg Glu Ile Arg Cys His Gln His Arg His Cys Glu Pro Asn

305 310 315 320

Gln Gly Leu Arg Val Lys Lys Glu Gly Thr Ala Glu Ser Asp Thr Val

325 330 335

Cys Thr Cys Lys Glu Gly Gln His Cys Thr Ser Lys Asp Cys Glu Ala

340 345 350

Cys Ala Gln His Thr Pro Cys Ile Pro Gly Phe Gly Val Met Glu Met

355 360 365

Ala Thr Glu Thr Thr Asp Thr Val Cys His Pro Cys Pro Val Gly Phe

370 375 380

Phe Ser Asn Gln Ser Ser Leu Phe Glu Lys Cys Tyr Pro Trp Thr Ser

385 390 395 400

Cys Glu Asp Lys Asn Leu Glu Val Leu Gln Lys Gly Thr Ser Gln Thr

405 410 415

Asn Val Ile Cys Gly Leu Lys Ser Arg Met Arg Ala Leu Leu Val Ile

420 425 430

Pro Val Val Met Gly Ile Leu Ile Thr Ile Phe Gly Val Phe Leu Tyr

435 440 445

Ile Lys Lys Val Val Lys Lys Pro Lys Asp Asn Glu Ile Leu Pro Pro

450 455 460

Ala Ala Arg Arg Gln Asp Pro Gln Glu Met Glu Asp Tyr Pro Gly His

465 470 475 480

Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro Val

485 490 495

Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln

500 505 510

Val Thr Asp Ser Ile Ala Leu Arg Pro Leu Val

515 520

Claims (61)

1. An expression cassette comprising a promoter operably coupled to a recombinant nucleic acid having first and second nucleic acid segments;

wherein the first nucleic acid segment encodes a chimeric protein having an extracellular portion of a TNF family ligand coupled to its corresponding TNF family receptor by a flexible linker; and is

Wherein the second nucleic acid segment encodes a Tumor Associated Antigen (TAA).

2. The expression cassette of claim 1, wherein the extracellular portion of the TNF family ligand is more N-terminal than the corresponding TNF family receptor on the chimeric protein.

3. The expression cassette of claim 2, wherein the TNF family ligand is CD40L, and wherein the TNF family receptor is CD 40.

4. The expression cassette of claim 3, wherein the recombinant nucleic acid further comprises a third nucleic acid segment encoding a leader peptide coupled to the N-terminus of the extracellular portion of CD 40L.

5. The expression cassette of claim 3 or 4, wherein the extracellular portion of CD40L is the human extracellular portion of CD 40L.

6. The expression cassette of any one of claims 3-5, wherein the flexible linker has between 4 and 50 amino acids, and optionally comprises a (GnS) x sequence.

7. The expression cassette of any of the preceding claims, wherein the TAA is selected from the group consisting of short-tail mutein, MUC1 and CEA.

8. The expression cassette of any of the preceding claims, wherein the TAA is a patient-specific and tumor-specific neoepitope.

9. The expression cassette of any one of the preceding claims, wherein the first and second nucleic acid segments are placed in the same reading frame and/or are separated by a 2A sequence.

10. The expression cassette of any one of claims 1-8, wherein the first and second nucleic acid segments are coupled via an IRES sequence.

11. The expression cassette of any of the preceding claims, further comprising a fourth nucleic acid segment encoding an immunostimulatory cytokine.

12. The expression cassette of claim 11, wherein the immunostimulatory cytokine is an IL-15 superagonist (ALT803) coupled to at least one of IL-7 and IL-21.

13. A virus comprising the expression cassette of any one of claims 1-12.

14. The virus of claim 13, wherein the virus is an adenovirus.

15. The virus of claim 14, wherein the adenovirus is AdV5[ E1-, E2b- ].

16. A method of treating a patient having a tumor, the method comprising administering the virus of any one of claims 13-15 to the patient.

17. The method of claim 16, further comprising administering a checkpoint inhibitor to the patient.

18. The method of claim 16 or 17, further comprising administering to the patient an IL-15 super agonist (ALT803) coupled to at least one of IL-7 and IL-21.

19. The method of any one of claims 16-18, further comprising co-administering a genetically modified bacterium or a genetically modified yeast as an adjuvant to the virus.

20. The method of claim 19, wherein the genetically modified bacteria express endotoxin at a level insufficient to induce CD-14 mediated sepsis.

21. The method of claim 20, wherein the genetically modified yeast belongs to a GI-400 series recombinant immunotherapeutic yeast strain.

22. A genetically modified immune cell comprising the expression cassette of any one of claims 1-12.

23. The genetically modified immune cell of claim 22, wherein the genetically modified immune cell is derived from a dendritic cell.

24. Use of the expression cassette of any one of claims 1-12, the virus of any one of claims 13-15, or the immune cell of claim 22 or 23 for the production of a pharmaceutical composition for treating a patient having cancer.

25. The use of claim 24, wherein the genetically modified immune cell is allogeneic to the patient and ex vivo expanded.

26. A method of producing an expression vector for enhancing immunotherapy, the method comprising:

constructing a recombinant nucleic acid having a sequence encoding: (a) a polyepitope operably linked to a first promoter to drive expression of the polyepitope, and (b) an adjuvant polypeptide operably linked to a second promoter to drive expression of the adjuvant polypeptide;

wherein the polyepitope comprises a trafficking element that directs the polyepitope to a subcellular location selected from the group consisting of: cytoplasm, recycled endosomes, sorted endosomes, lysosomes, and extracellular membranes, or wherein the trafficking element directs the polyepitope to the extracellular space; and is

Wherein the polyepitope comprises a plurality of filtered new epitope sequences.

27. The method of claim 26, wherein the adjuvant polypeptide is calreticulin or HMGB 1.

28. The method of claim 26 or 27, wherein at least one of the first and second promoters is a constitutive promoter.

29. The method of claim 26 or 27, wherein at least one of the first and second promoters is an inducible promoter.

30. The method of claim 29, wherein the promoter is inducible by hypoxia, IFN- γ or IL-8.

31. The method of any one of claims 26-30, wherein the transport element is selected from the group consisting of: cleavable ubiquitin, non-cleavable ubiquitin, CD1b leader, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

32. The method of any one of claims 26-31, wherein the filtered neoepitope sequences are filtered by comparing tumors of the same patient to matched normalcy, and optionally wherein the filtered neoepitope sequences are filtered to have a binding affinity to MHC complexes equal to or less than 200 nM.

33. The method of any one of claims 26-32, wherein the filtered neo-epitope sequences have an arrangement within the polyepitope such that the polyepitope has a likelihood of presence of a hydrophobic sequence or signal peptide, and/or has a hydrophobic sequence or signal peptide intensity below a predetermined threshold.

34. The method of any one of claims 26-33, wherein the filtered neo-epitopes bind to MHC-I, and wherein the trafficking element directs the polyepitope to the cytoplasm or proteasome.

35. The method of any one of claims 26-33, wherein the filtered neoepitope sequences bind to MHC-I, and wherein the trafficking element directs the polyepitope to a recirculating endosome, sorting endosome, or lysosome.

36. The method of any one of claims 26-33, wherein the filtered neoepitope sequences bind to MHC-II, and wherein the trafficking element directs the polyepitope to a recirculating endosome, sorting endosome, or lysosome.

37. The method of any one of claims 26-36, wherein the recombinant nucleic acid further comprises a sequence encoding a second polyepitope, wherein the second polyepitope comprises a second trafficking element that directs the second polyepitope to a different subcellular location, and wherein the second polyepitope comprises a second plurality of filtered neo-epitope sequences.

38. The method of claim 37, wherein at least some of the plurality of filtered new sequence of table bits and some of the second plurality of filtered new sequence of table bits are the same.

39. The method of any one of claims 26-38, wherein the recombinant nucleic acid further comprises a sequence encoding at least one of a co-stimulatory molecule, an immunostimulatory cytokine, and a protein that interferes with or down-regulates checkpoint inhibition.

40. The method of claim 39, wherein the co-stimulatory molecule is selected from the group consisting of: OX40L, 4-1BBL, CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1 and LFA 3.

41. The method of claim 39, wherein the immunostimulatory cytokine is selected from the group consisting of: IL-2, IL-12, IL-15 superagonists (ALT803), IL-21, IPS1 and LMP 1.

42. The method of claim 39, wherein the interfering protein is an antibody or antagonist to CTLA-4, PD-1, TIM1 receptor, 2B4 or CD 160.

43. The method of any one of claims 26-42, wherein the transport element is selected from the group consisting of: cleavable ubiquitin, non-cleavable ubiquitin, CD1b leader, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

44. The method of any one of claims 26-43, wherein the expression vector is selected from the group consisting of: adenovirus expression vectors, yeast expression vectors, and bacterial expression vectors deleted for the E1 and E2b genes.

45. A method of improving the immune response to cancer immunotherapy in an individual having a tumor, the method comprising:

administering a cancer vaccine composition to the tumor; and is

Substantially simultaneously co-administering an adjuvant polypeptide, ATP, or ATP analog to the tumor.

46. The method of claim 45, wherein the cancer vaccine composition comprises a recombinant adenovirus, a recombinant yeast, or a recombinant bacterium.

47. The method of claim 45 or 46, wherein the cancer vaccine composition comprises or encodes a tumor neoepitope of the patient.

48. The method of any one of claims 45-47, wherein the cancer vaccine composition is administered directly to the tumor.

49. The method of any one of claims 45-48, wherein the adjuvant polypeptide is calreticulin or HMGB 1.

50. The method of any one of claims 45-49, wherein the adjuvant is a non-hydrolyzable ATP analog.

51. The method of any one of claims 45-50, wherein the adjuvant polypeptide, ATP, or ATP analog is injected into the tumor.

52. A genetically engineered activated Dendritic Cell (DC) prepared by a method comprising:

infecting a tumor cell with a recombinant nucleic acid having first and second nucleic acid segments;

wherein the first nucleic acid segment encodes a chimeric protein having an extracellular portion of CD40 coupled to CD40L through a flexible linker; and is

Wherein the second nucleic acid segment encodes a tumor associated antigen.

53. The DC of claim 52, further comprising a recombinant nucleic acid encoding an antibody secreting portion that affects a tumor microenvironment.

54. The DC of claim 52 or 53, wherein the antibody secreting portion comprises one or more of: PD1, CTLA4 and TGF beta trap and IL-8.

55. A method of treating a tumor in a patient in need thereof, the method comprising administering to the patient a composition comprising the DC of any one of claims 52-54.

56. The method of claim 55, wherein the administration is intratumoral.

57. The method of claim 55, wherein the administration is topical.

58. The method of claim 55, wherein the administration is by injection into the tissue of the patient.

59. The method of claim 55, wherein the administration is by inhalation.

60. The method of claim 55, wherein the administration is intrathecal.

61. The method of any one of claims 55-60, wherein the tumor is a bladder tumor, brain tumor, skin tumor, liver cancer, breast cancer, pancreatic cancer, and/or lung cancer.

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